Deamidation disrupts native and transient contacts to weaken the interaction between UBC13 and RING-finger E3 ligases
Several bacterial pathogens secrete effector proteins that inhibit or co-opt the Ubiquitin ( Ub ) pathway to suppress the immune response of the host cell ( Ashida et al . , 2014 ) . The human pathogenic bacteria Shigella flexneri inactivates the host inflammatory Nf-κB signaling , responsible for inducing inflammatory cytokine responses during pathogen invasion ( Sanada et al . , 2012 ) . In the early events of interleukin-dependent activation of Nf-κB signaling , the Ub-conjugating enzyme UBC13 , and the Ub-ligase TRAF6 function together to synthesize both unanchored polyubiquitin chains , and anchored polyubiquitin chains on TRAF6 and its substrate NEMO ( Chen , 2005 ) . These chains serve as a scaffold to bring together TAK1/2 and IKK kinases , eventually leading to phosphorylation and activation of the IKK kinases , IκB degradation and nuclear translocation of transcription factor Nf-κB . To inactivate Nf-κB signaling , Shigella flexneri secretes a Type III effector called OspI , which functions as a deamidase ( Sanada et al . , 2012 ) . OspI specifically targets a glutamine residue in UBC13 and converts it to glutamate ( Q100E ) . Ubiquitination reactions of TRAF6 either with UBC13 in the presence of OspI or with mutant Q100E-UBC13 ( dUBC13 ) show a significant drop in polyubiquitination activity ( Sanada et al . , 2012 ) . However , the mechanism underlying inhibition of polyubiquitination by deamidation of UBC13 remains unclear . Ubiquitination is a eukaryotic post-translational modification ( Komander and Rape , 2012 ) , wherein the last glycine residue in the C-terminal tail of Ubiquitin ( Ub ) is activated and covalently attached to a substrate lysine residue . Ubiquitination involves three steps: an initial activation and thioester conjugation by the Ubiquitin-activating enzyme ( E1 ) , followed by the thioester conjugation to the Ubiquitin-conjugating ( E2 ) enzymes , and a final step in which the Ub is covalently attached to the substrate amino group . The last step is typically catalyzed by a class of Ubiquitin ligases ( E3 ) , which contain either a RING ( Really Interesting New Gene ) -finger domain , U-box domain or HECT domain ( Metzger et al . , 2012 ) . The RING-finger/U-box domain stabilizes a catalytic-closed conformation of the flexible E2 ~Ub species and drastically enhances the rate of Ub conjugation to substrates ( Dou et al . , 2012; Plechanovová et al . , 2012; Pruneda et al . , 2012 ) . The E2 UBC13 functions with several RING-finger E3s like TRAF6 to synthesize K63-linked poly-Ub chains that function to activate DNA repair or immune response ( Fukushima et al . , 2007 ) . Apart from the E3s , UBC13 also binds a co-factor MMS2 , which does not activate UBC13 but maintains the linkage specificity of the poly-Ub chains synthesized by UBC13 ( Branigan et al . , 2015 ) . In this study , we have investigated the mechanisms underlying the inactivation of UBC13 upon deamidation using NMR spectroscopy , molecular dynamics ( MD ) simulations , and in-vitro ubiquitination assays . We report that deamidation weakens the non-covalent interaction of UBC13 with RING-finger domain of TRAF6 ( TRAF6RING ) , without perturbing UBC13 structure or the intrinsic enzymatic activity of UBC13 . However , the underlying cause of reduced interaction is nonintuitive since Q100 is in the vicinity of UBC13/TRAF6RING interface but does not form any contact with the TRAF6RING . Further studies showed that deamidation disrupts the interaction between UBC13 and TRAF6RING by three mechanisms: i ) A new intramolecular R14/E100 salt-bridge appears in dUBC13 , which competes with a critical intermolecular salt-bridge in the native complex , ii ) the salt-bridge competition also perturbs the UBC13/TRAF6RING transient complexes to inhibit association , and iii ) transient repulsion between the negatively charged E100 and the negatively charged interface of TRAF6RING reduces association between UBC13 and TRAF6RING . The effect of each mechanism on the binding was confirmed by binding studies using appropriate substitutions in either UBC13 or TRAF6RING . The impact of deamidation on transient interactions was also observed using another RING domain from RNF38 , indicating that the mechanism could be ubiquitous for UBC13/RING complexes . Our study highlights that deamidation of residues that do not directly participate in the E2/E3 interaction but are close to the interface can effectively modulate the interaction by perturbing the native and transient intermolecular contacts . The mechanism of regulating protein-protein transient interactions by post-translational modifications could be at play in other quintessential signaling pathways .
Two different constructs of TRAF6 was used in this study . The isolated RING domain TRAF6RING ( aa: 50–124 ) was a shorter construct . The longer construct included the RING domain and three ZF domains ( TRAF6RZ3 , aa: 50–211 ) . TRAF6RING interacts with UBC13 and with the donor Ub in the UBC13 ~Ub conjugate , while the interaction of ZF domains with donor Ub further stabilized the complex ( Middleton et al . , 2017 ) . dUBC13 had reduced polyubiquitination activity than UBC13 , either with longer TRAF6RZ3 or with shorter TRAF6RING ( Figure 1A and B ) . Deamidation could have some allosteric effect at the active site or Ub-binding site , which may deactivate UBC13 . However , a comparison of the extent of polyubiquitination by the UBC13/MMS2 heterodimer in the absence of E3 was similar between UBC13 and dUBC13 , indicating that E2 activity is not altered upon deamidation ( Figure 1C ) . Deamidation could misfold UBC13 or inhibit the interaction between UBC13 and TRAF6RING . These possibilities were examined by studying the impact of deamidation on the structure of UBC13 and its interaction with TRAF6RING . 15N-labeled UBC13 , dUBC13 , and unlabelled TRAF6RING were expressed in E . coli and purified . Unlabelled TRAF6RING was titrated into a sample of 15N-UBC13 , and the binding was detected by 15N-edited Heteronuclear Single Quantum Coherence ( HSQC ) NMR experiments ( Figure 1—figure supplement 1 ) . Perturbations due to the altered chemical environment upon ligand binding induce changes in the chemical shift of the backbone amide resonances . The chemical shift perturbations ( CSP ) plotted in Figure 1D shows that the significant perturbations in UBC13 occur in the α1-helix , and the loop between α3-10 and α2-helix , which is the canonical UBC13 interface in the complex ( Figure 1G and H ) . The resonance shifts can be plotted against the ligand: protein concentration , and fit to yield the dissociation constant ( Kd ) of interaction . The peak shifts in UBC13 titration spectra were fitted to yield a Kd of 0 . 39 ( ±0 . 04 ) mM ( Figure 1I and Figure 1—figure supplement 1B ) . Backbone amide chemical shifts can report if the protein’s fold is affected by the substitution/modification of a residue . The backbone chemical shifts of UBC13 and dUBC13 resonances were compared . The amide CSPs between UBC13 and dUBC13 indicated that apart from the residues immediately next to Q100 in sequence , only R14 and L15 of the N-terminal α1-helix are affected upon Q100E substitution ( Figure 1—figure supplement 2A ) . The absence of major CSPs in the rest of dUBC13 indicated that deamidation did not change the fold of UBC13 . Moreover , analysis of the backbone and Cβ chemical shifts in dUBC13 confirmed that the secondary structure was unperturbed ( Figure 1—figure supplement 2B ) . Unlabelled TRAF6RING was then titrated into a sample of 15N-dUBC13 , and the binding was detected by 15N-edited HSQC experiments ( Figure 1—figure supplement 3 ) . Negligible peak shifts were detected in dUBC13 even when the TRAF6RING was titrated up to 6-fold higher than dUBC13 , indicating that TRAF6RING does not bind dUBC13 ( Figure 1E–H ) . A rough estimate of the Kd was obtained by comparing the CSPs at the UBC13 interface at the same protein: ligand concentration between UBC13 and dUBC13 , which suggested the Kd of 6 . 34 ( ±2 . 6 ) mM . If the reduced affinity is precisely due to the negative charge at residue 100 , then substitution of Q100 with a neutral amino acid should not affect the affinity of the complex . TRAF6RING was titrated to Q100A-UBC13 , and the measured Kd was 0 . 54 ( ±0 . 07 ) mM ( Figure 1I ) , which is similar to UBC13/TRAF6RING . However , when Q100 was substituted with another acidic residue aspartate ( D ) , the affinity dropped significantly ( Kd ~4 . 6 ( ±1 . 8 ) ) mM , Figure 1I ) , confirming that substitution at Q100 with a negatively charged residue inhibited the interaction between UBC13 and TRAF6RING . Altogether , deamidation inhibited the binding of TRAF6RING to UBC13 , but neither changed the fold of UBC13 nor its intrinsic E2 activity . In the UBC13/TRAF6RING native complex structure , Q100 does not form any direct contact with TRAF6RING ( Figure 2A ) . Hence , the mechanism underlying reduced binding due to deamidation of Q100 is nonintuitive . The surface electrostatic potentials calculated for UBC13 and TRAF6RING indicated charge complementarity at the interface ( Figure 3A ) . The UBC13 interface was positively charged , while the TRAF6RING was negatively charged . Notably , Q100 lies in the vicinity of a network of electrostatic interactions involving helix-1 of UBC13 ( R6 and R14 ) and the first zinc-coordination motif ( D57 and E69 ) of the TRAF6RING ( Figure 2A ) . Possibly , the introduction of an additional negative charge in UBC13 upon deamidation could perturb the interfacial electrostatic contacts to disrupt the interaction . A conventional MD simulation ( 200 ns ) of free dUBC13 was performed to investigate if deamidation alters any electrostatic interactions within UBC13 . Interestingly , an intramolecular salt-bridge was observed between R14 and E100 in dUBC13 ( Figure 2B ) , which correlates well with the backbone chemical shift perturbations observed at R14 upon deamidation ( Figure 1—figure supplement 2A ) . The new salt-bridge involving R14 and E100 in dUBC13 was further investigated by NMR . 15N-edited HSQCs of the Arginine Nε-Hε groups were collected for both UBC13 and dUBC13 , which showed shifted resonances for the arginine sidechains ( R7 , R14 , and R102 ) around residue E100 , indicating that these sidechain conformations are perturbed ( Figure 2C ) . Unfortunately , the 1Hη atoms are invisible in the NMR spectra due to chemical exchange with the solvent at physiological pH , and millisecond timescale rotations around the Cζ-Nη/Cζ-Nε bonds ( Figure 2D ) . Hence , detection of arginine sidechain-mediated interactions ( hydrogen bonds and salt-bridges ) through 1Hη atoms is difficult . However , such interactions can be inferred from the reduced mobility of their sidechains observed in 15Nη/ε ( F1 ) -13Cζ ( F2 ) correlation spectra ( Yoshimura et al . , 2017 ) . For example , R85 forms salt-bridge with D81 in UBC13 , which reduces the sidechain mobility of R85 sidechain and gives rise to two separate resonances for R85 Nη atoms in the 15Nε/η-13Cζ correlation spectra ( Figure 2E ) . The side-chains of free arginines rotate faster and their corresponding Nη atoms have a single averaged resonance in the spectra ( Figure 2E ) . The R14 Nη atoms had two separate resonances in dUBC13 but not UBC13 ( Figure 2F , expanded in 2G ) , implying that R14 forms a new intramolecular salt-bridge in dUBC13 , consistent with the MD simulations . The salt-bridge persisted in the Q100D-UBC13 ( Figure 2—figure supplement 1 ) , correlating well with its reduced affinity for TRAF6RING . At the interface of UBC13/TRAF6RING , R14 of UBC13 forms an intermolecular salt-bridge with E69 of TRAF6RING ( Figure 2A ) . The deamidation-induced intramolecular R14/E100 salt-bridge may interfere with the intermolecular R14/E69 salt-bridge to destabilize the complex . Conventional MD simulations were performed with both wild type ( wt ) and deamidated complex to test this hypothesis . The stability of the complex was determined by the rmsd of TRAF6RING with respect to the crystal structure , where higher rmsd implied instability . The wt complex was stable throughout the 200 ns trajectory with low rmsd ( Figure 2—figure supplement 2A ) . The deamidated complex was unstable for a significant duration of 75 ns , wherein the rmsd of TRAF6RING increased substantially ( Figure 2—figure supplement 2B ) . Five interfacial contacts were chosen as reporters of the interaction between UBC13 and TRAF6RING ( Figure 2—figure supplement 2 ) . Among them , three were electrostatic ( Figure 2A and Figure 2—figure supplement 2A ) , and the rest were hydrophobic ( Figure 2—figure supplement 2D ) . The other interfacial contacts were mostly unstable in simulations and were omitted from our analysis ( Figure 2—figure supplement 2G and H ) . No major instability was observed in the hydrophobic contacts between UBC13 and dUBC13 ( Figure 2—figure supplement 2E and F ) . However , concurrent to the high rmsd in dUBC13 complex , the R14/E69 intermolecular salt-bridge disrupted and the R14/E100 intramolecular salt-bridge formed , suggesting that salt-bridge competition might destabilize the native dUBC13 complex ( Figure 2—figure supplement 2B , Video 1 ) . The occupancy of the R14/E69 intermolecular salt-bridge dropped from 99% in the wt complex to 40% in the dUBC13 complex ( Supplementary file 1-table S1 ) . The Umbrella Sampling ( US ) method was used to estimate the stability of the deamidated complex ( Figure 3 ) . The US method quantifies the energy required ( ΔGPMF ) to form the native complex . An unstable complex is associated with higher energy . As a control , US simulations were performed on the Barnase/Barstar complex ( Table 1 and Supplementary file 1-table S2 , Figure 3—figure supplement 1A ) . The PMF profile and ΔGPMF of the complex are consistent with its high stability ( Hoefling and Gottschalk , 2010; Wang et al . , 2010 ) . The US simulations were then carried out for wt , dUBC13 , Q100A-UBC13 , and R14A-UBC13 complexes ( Table 1 , Supplementary file 1-table S2 , Figure 3—figure supplement 1B ) . The PMF profile of wt complex decreased sharply by 2 . 0 kcal/mol at 3 . 5 nm and subsequently plateaued ( Figure 3B ) . The sharp drop in the PMF profile of the wt complex was due to attractive electrostatic interactions , which were absent in the other complexes ( Figure 3—figure supplement 1C and D ) . In contrast , ΔGPMF of dUBC13 complex gradually reduced by only 0 . 7 kcal/mol , suggesting that deamidation reduces the stability of the complex . The difference in ΔGPMF between the deamidated and wt complex , ΔΔGPMF = 1 . 37 kcal/mol was of the same order as the decrease in binding energy observed by NMR titrations , ΔΔG = 1 . 69 kcal/mol ( Figure 1I ) . The ΔGPMF of Q100A-UBC13/TRAF6RING is similar to the wt complex ( Table 1 ) . The stability of the intermolecular contacts was compared between UBC13 and dUBC13 complexes at the native US window . While the R14/E69 salt-bridge was stable throughout in the wt complex , it disrupted in the dUBC13 complex ( Figure 3C and D , Supplementary file 1-table S5 ) . The Coulombic Interaction Energy ( CIE ) is the sum energy of all the electrostatic interactions . Lower CIE values reflect attractive electrostatic interactions . The mean CIE was significantly higher in dUBC13 complex , suggesting that deamidation primarily inhibited electrostatic interactions ( Figure 3F ) . The disruption of R14/E69 salt-bridge temporally correlates well with higher CIE and higher rmsd of TRAF6RING ( Figure 3E and G ) . Interestingly , when the US simulations were repeated with R14A-UBC13 , the ΔGPMF was insignificant , indicating that R14-mediated interactions are essential for the binding ( Figure 3B ) . Overall , the conventional MD and US simulations indicate that salt-bridge competition may sequester R14 away from the interface and reduce the binding energy of the complex . Steered MD ( SMD ) simulations were performed to capture the effects of deamidation on the dissociation of the UBC13 and dUBC13 complexes . If a complex is unstable , less force and work will be required to dissociate it . Compared to the wt complex , less force and work were required to dissociate the dUBC13 complex , indicating reduced stability due to deamidation ( Figure 4A–C , Table 2 and Supplementary file 1-table S3 ) . As TRAF6RING starts to dissociate , the intramolecular R14/E100 salt-bridge competes with the intermolecular R14/E69 salt-bridge and reduces its strength ( Figure 4D , Figure 4—figure supplements 1–2 and Supplementary file 1-table S4 ) . The R14A-UBC13 complex , where the intermolecular R14/E69 salt-bridge is absent , was also less stable compared to the wt complex ( Table 2 ) . The order in which the contacts disrupted during dissociation was compared in a typical SMD trajectory for both UBC13 and dUBC13 complexes ( Figure 5A and Videos 2 and 3 ) . In the wt complex , whereas the other contacts disrupted early , the R14/E69 intermolecular salt-bridge persisted till the complex dissociated completely ( Figure 5A–5C and Figure 5—figure supplement 1A ) . Since the intermolecular salt-bridge was stable for a long duration in both the conventional and SMD simulations , it could be critical for the wt complex . However , in the trajectory of the dUBC13 complex , the intermolecular salt-bridge disrupted prematurely , and the intramolecular salt-bridge formed simultaneously ( Figure 5D–5F and Figure 5—figure supplement 1B ) . Collectively , SMD suggested that R14/E69 formed a critical interfacial salt-bridge , and as the molecules start to dissociate , the R14/E69 and R14/E100 salt-bridges compete . The competition may contribute to the reduced stability and enhanced dissociation of the dUBC13 complex . A native protein complex forms through an intermediate species commonly referred to as the transient/encounter complex ensemble ( Schreiber et al . , 2009 ) . The transient complexes are formed by long-range electrostatic interactions and have greater oriental freedom compared to the native complex ( Tang et al . , 2006 ) . The effect of negatively charged E100 on the transient complex was estimated based on transient complex theory ( Alsallaq and Zhou , 2008 ) . The method uses the native complex structure to compute its association rate constant ( kon ) from the electrostatic free energy ( ΔGel ) of the transient complex ensemble . Position of individual atoms within each input structure remained fixed in transient-complex theory calculations , and hence , the competition between salt-bridges was absent . Therefore , any difference between wt and deamidated complex were solely due to transient repulsion between E100 and the acidic residues of TRAF6RING . The association rate constant of the wt complex reduced at high salt compared to low salt , confirming that electrostatic interactions are important for the association of wt transient complex ( Table 3 ) . The association rate constant reduced by three-fold for dUBC13/TRAF6RING at low salt , indicating that deamidation reduces the association of the transient complex . The difference between wt and dUBC13 complex is nominal at high salt , where the repulsive effects of E100 may be screened . Multiple US windows also indicated repulsive transient interactions between E100 and TRAF6RING ( Figure 6 , Figure 6—figure supplement 1A ) . The molecular details of the repulsive effect could be observed in a typical US window ( Figure 6 ) . In the wt complex , a transient attractive interaction was observed between R14 in UBC13 and D57 in TRAF6RING ( Figure 6A and C , Supplementary file 1-table S5 ) . The wt complex was stable with low rmsd and low CIE values ( Figure 6B and D ) . However , the R14/D57 interaction was absent in the dUBC13 complex , and it dissociated prematurely ( Figure 6B–D , Supplementary file 1-table S5 ) . In the initial timepoints of this trajectory , R14 and E100 simultaneously interacted with D57 ( Figure 6C and E ) . While the R14/D57 interaction was attractive , the E100/D57 interaction was repulsive . The repulsive interaction probably destabilized the attractive interaction , and it disrupted soon after . The complex dissociated subsequently ( Figure 6B ) . The instability of deamidated complex was also evident in other US windows ( Figure 6—figure supplement 1B–E ) . Overall , electrostatic repulsion between E100 and the negatively charged interface of TRAF6RING was observed in the dUBC13 transient complexes , which could destabilize it . When the transient complex theory calculations were repeated for the R14A-UBC13 complex , the kon dropped significantly by six-fold at low salt ( Table 3 ) . Since repulsion due to E100 is absent in R14A-UBC13 , the drop in kon suggests that R14 also forms transient contacts with TRAF6 , which are important for the binding . US simulations also suggested that R14-mediated non-native transient interactions promote the association of the wt complex ( Figure 6A ) . Unbiased-association simulations were performed on the complexes to confirm this observation . From these simulations , the probability of association and stability of native-like transient complexes were assessed ( Table 4 ) . Similar to Transcomp calculations ( Table 3 ) , the native-like association of the wt complex was 3-fold higher at low salt than high salt , confirming that formation of the transient complex was dependent on the long-range electrostatic interactions . The association and stability dropped by 2 . 3 fold in the dUBC13 complex at low salt , suggesting that deamidation reduces the formation of the native-like transient complex . The ensemble of transient complexes was compared between UBC13 and dUBC13 ( Figure 7 ) . The distribution of TRAF6RING positions around the helix 1 of UBC13 indicates that the TRAF6RING positions are tightly clustered around the UBC13 binding interface ( Figure 7A ) . The projections of the transient complexes showed that a cluster indeed coincided with the final native complex . The positions of TRAF6RING are dispersed when it interacts with dUBC13 , and none of the clusters coincide with the native complex ( Figure 7B ) . Several TRAF6RING clusters around R14 were absent in the dUBC13 complex , indicating that the transient contacts between R14 and TRAF6RING are absent in dUBC13 ( Figure 7C and D ) . The energy landscape correlating the free-energy with rmsd of TRAF6RING , and the minimum distance between interfaces shows multiple low-energy transient intermediates were present in the association pathway , which converged to the native complex ( Figure 7—figure supplement 1A ) . In contrast , a few transient intermediates were present in the dUBC13 complex , and none existed in the native-like conformation ( Figure 7—figure supplement 1A ) . The association trajectories were further analyzed to detect native-like complexes , which identified one trajectory at low salt for the wt complex ( Figure 7—figure supplement 2A , Figure 8A ) . Here , TRAF6RING associated initially with UBC13 via the R6/D57 and R14/E69 salt-bridges ( Figure 8B , Video 4 ) . Subsequently , the other contacts formed , leading to a stable native complex ( Figure 8—figure supplement 1A ) . No native complex was detected for the dUBC13 complex at low salt conditions ( Figure 7—figure supplement 2B ) . At higher salt , where deamidation effects are potentially screened , a native-like complex was momentarily observed ( Figure 7—figure supplement 2B and Figure 8C , Video 5 ) . Here , a non-native transient R6/E69 salt-bridge and the S96/P106 hydrogen bond formed for a short period . However , the intramolecular R14/E100 salt-bridge was strong , whereas the intermolecular R14/E69 salt-bridge and other contacts were unstable ( Figure 8C and D , and Figure 8—figure supplement 1B ) . To summarize , unbiased association simulations indicate that the critical transient contacts formed by R14 were absent in the deamidated complex , presumably due to the salt-bridge competition . The MD simulations suggested that deamidation destabilized both the native and transient complexes . For simplicity , we assumed that the effect of salt-bridge competition on the transient and native complexes , and the repulsive effect of E100 on transient complexes are mutually exclusive . The difference in binding energy due to deamidation ( ΔΔGd ) is then ( 1 ) ΔΔGd=ΔΔGNSB+ΔΔGTSB+ΔΔGTrep , where ΔΔGNSB is the difference in binding energy due to salt-bridge competition in the native complex , ΔΔGTSB is due to salt-bridge competition in the transient complex , and ΔΔGTrep is the repulsive effect of E100 on the transient complex . NMR titrations were carried out using variants of UBC13 and TRAF6RING to validate the effect of each mechanism on the binding energy of the complex . E69A-TRAF6RING removed the acceptor of the intermolecular salt-bridge and mimicked the effect of salt-bridge competition in the native complex ( ΔΔGNSB ) . When 15N-UBC13 was titrated with E69A-TRAF6RING , the Kd increased , and binding energy decreased ( Figure 9A ) . The drop in binding energy compared to the wt complex gave the ΔΔGNSB = 0 . 82 kcal/mol . The R14A-UBC13 substitution mimicked the salt-bridge competition in both the native and the transient complexes , that is the drop in binding energy was ΔΔGNSB + ΔΔGTSB . The titration of R14A-UBC13 with TRAF6RING determined a drop in the binding energy by 1 . 45 kcal/mol ( Figure 9A ) . Given the value of ΔΔGNSB was determined above , ΔΔGTSB = 0 . 63 kcal/mol . Deamidation reduced the binding energy by 1 . 69 kcal/mol ( ΔΔGd , Figure 1I ) . Since ΔΔGNSB + ΔΔGTSB = 1 . 45 kcal/mol , ΔΔGTrep = 0 . 24 kcal/mol ( Equation 1 ) . The value of ΔΔGTrep agreed well with the repulsive effect calculated by transient complex theory calculations , ΔΔGel = ΔGelUBC13 - ΔGeldUBC13 = 0 . 34 kcal mol−1 ( Table 3 ) . Overall , titrations studies indicated that all three mechanisms have distinct contributions to the binding between UBC13 and TRAF6RING . Analysis of the UBC13/RING structures shows that R14-mediated salt-bridges are common in these complexes ( Figure 9—figure supplement 1 ) . However , in a few complexes like UBC13/RNF4 and UBC13/RNF8 , the R14 mediated salt-bridge was absent . A typical RING domain from the E3 RNF38 ( RNF38RING , aa: 387–465 ) was chosen to study the effect of UBC13 deamidation in such cases . RNF38RING can activate UBC13 to synthesize polyubiquitin chains ( Figure 9B ) . Similar to TRAF6 , the RNF38RING fails to activate dUBC13 ( Figure 9B ) . Like RNF4 and RNF8 , RNF38RING lacks a negatively charged residue at the region corresponding to E69 in TRAF6RING ( Figure 9—figure supplement 2A–C ) . However , there are several acidic residues in the vicinity , similar to TRAF6RING , which can form non-native transient interactions with UBC13 ( Figure 9—figure supplement 2B and C ) . NMR titrations measured the effect of deamidation on the interaction between UBC13 and RNF38RING . When RNF38RING domain was titrated to 15N-UBC13 ( Figure 9C ) , major CSPs were observed in the α1-helix , β3-β4 loop , and the loop between α3-10 and α1-helix ( Figure 9D and E ) . The Kd value of UBC13/RNF38RING interaction was 0 . 04 mM ( Figure 9I ) . Considerable peaks shifts were also detected when RNF38RING was titrated to 15N-dUBC13 ( Figure 9F ) . However , the CSPs in dUBC13 were reduced compared to UBC13 at the same protein: ligand stoichiometry , indicating reduced affinity ( Figure 9F–H ) . Fitting of peak shifts against ligand: protein ratio yielded the Kd to be 0 . 25 mM , which corresponds to a 6-fold drop in affinity . This could be due to the combination of the salt-bridge competition and repulsive interactions . The backbone chemical shifts of RNF38RING resonances were assigned using 13C , 15N labeled RNF38RING sample , and standard triple resonance NMR experiments . The interface of RNF38RING was mapped by titrating unlabeled UBC13 to 15N-labeled RNF38RING ( Figure 9—figure supplement 2D ) . Using the CSPs of UBC13 and RNF38RING , an NMR-data driven structural model of UBC13/RNF38RING was determined by HADDOCK ( Figure 9—figure supplement 2E ) . The structure showed that in the absence of a negative charged salt-bridge acceptor , R14 makes van der Waals contacts with M417 ( Figure 9—figure supplement 2F ) . The R14A substitution reduces the binding energy of the native complex by 0 . 2 kcal/mol ( Vangone and Bonvin , 2015 ) , indicating it is not a hot-spot in this complex . The titration was repeated with R14A-UBC13 , which mimics the R14 sequestration by salt-bridge competition . The Kd of R14A-UBC13/RNF38RING showed a 2 . 5-fold increase and reduced binding energy by 0 . 6 kcal/mol . Since the effect of R14A substitution on the native complex is 0 . 2 kcal/mol , the effect on the transient complex is 0 . 4 kcal/mol . Deamidation reduces the binding energy by 1 . 1 kcal/mol ( Figure 9I ) . Given the salt-bridge competition reduces binding energy by 0 . 6 kcal/mol , the repulsive effect of E100 on the transient complex is 0 . 5 kcal/mol . Overall , the effect of deamidation on perturbing the ensemble of transient complexes was verified in another RING domain , which indicated that the mechanism could be ubiquitous in UBC13/RING complexes . If the RING domains bind weakly to dUBC13 , their ability to activate the dUBC13 ~Ub conjugate should be compromised , which was tested by binding and activity assays . First , the conjugation of donor Ub to the UBC13 and dUBC13 were compared by an in-vitro conjugation reaction . Both the UBC13 and dUBC13 conjugated with the donor Ub with similar efficiency , indicating that deamidation does not hamper E2 ~Ub conjugation ( Figure 10A and Figure 10—figure supplement 1 ) . In the UBC13 ~Ub/TRAF6RZ3 complex , the RING domain and ZF1 domain makes additional contacts with donor Ub to stabilize the complex further . These contacts could rescue the loss in affinity between UBC13 and RING domain upon deamidation . Hence , the binding of UBC13 ~Ub and dUBC13 ~Ub to TRAF6RZ3 was compared . While UBC13 ~Ub conjugate bound to TRAF6RZ3 , dUBC13 ~Ub conjugate failed to do so ( Figure 10B and Figure 10—figure supplement 1 ) . In a similar experiment , UBC13 ~Ub but not dUBC13 ~Ub bound to RNF38RING ( Figure 10C ) . Altogether , the binding experiments confirmed that secondary interactions between Ub/RING or Ub/ZF1 could not compensate for the loss of primary interaction between UBC13 and RING . The activation of UBC13 ~ Ub conjugates by the RING domains were measured by the kinetics of single-round Ub discharge from the UBC13 ~ Ub . UBC13 ( or dUBC13 ) was first conjugated with K63A-Ub . Then the conjugation reaction was quenched , followed by the addition of Mms2 , RING domains , and free Lysine . TRAF6RING could effectively discharge Ub from UBC13 ~ Ub , but not from dUBC13 ~ Ub ( Figure 10D , G , H ) . Similarly , RNF38RING could discharge Ub from UBC13 ~ Ub but not from dUBC13 ~ Ub ( Figure 10E , I , J ) . When the concentration of the RNF38RING was increased by five-fold to compensate for the reduced affinity of dUBC13/RING complex , the rate of Ub discharge improved , which confirmed that the cause of the inefficient discharge is the reduced affinity of dUBC13/RING interaction ( Figure 10K and F ) . The lysine of the acceptor Ub at the growing end of a poly-Ub chain ( either unanchored or anchored on a substrate ) , attacks the E2 ~Ub active site to transfer the donor Ub . The kinetics of single-round di-Ubiquitin ( Ub2 ) synthesis was monitored to investigate if the deamidation of UBC13 affects the activity of UBC13 and its interaction with acceptor lysine at the active site . Here the lysine of the acceptor Ub mimics the acceptor lysine of growing poly-Ub chain . UBC13 ( or dUBC13 ) was conjugated with donor K63A-Ub . Then Mms2 , RNF38RING and acceptor D77-Ub was added in the reaction mix , which was then monitored over time to detect the synthesis of Ub2 . The rate of Ub2 synthesis was rapid for UBC13 but slow for dUBC13 ( Figure 10L ) . Kinetic analysis indicated that the reaction rate for the dUBC13/RING complex is similar to the rate of UBC13 in the absence of RING ( Figure 10M and Figure 10—figure supplement 2 ) , indicating that the catalytic effect of RING is absent in the dUBC13/RING complex . A comparison of the kinetic parameters between UBC13 and dUBC13 indicated that deamidation does not significantly affect the Km of the substrate , but drastically affects the Kcat of the reaction ( Figure 10N and Figure 10—figure supplement 2 ) . This confirms that the area around the active site is not perturbed by deamidation , as was observed by NMR ( Figure 1—figure supplement 2 ) . Furthermore , in the absence of a RING domain , the Kcat of UBC13 and dUBC13 is similar , confirming that the catalytic activity of UBC13 is unaffected upon deamidation , similar to the observation of Figure 1C . However , unlike UBC13 , the Kcat of dUBC13 does not increase in the presence of RING , confirming that RING domains fail to bind and activate the dUBC13 ~Ub conjugate .
Protein deamidation can have significant functional consequences for innate immune signaling ( Zhao et al . , 2016 ) , which justifies its emergence as a powerful tool employed by pathogenic bacteria to suppress the immune response ( Washington et al . , 2013 ) . However , little is known about the molecular mechanism underlying deactivation of immune signaling by deamidation . The Shigella flexneri effector OspI suppresses the inflammatory response ( NF-κB pathway ) via deamidation of UBC13 at Q100 . By an unknown mechanism , deamidation of Q100 to E100 inhibits the synthesis of K63-linked polyubiquitin chains by the UBC13/TRAF6 complex , preventing the downstream activation of NF-κB pathway . Q100 is present at the vicinity of UBC13/TRAF6 interface but does not form direct contact with TRAF6 . We report that deamidation neither alters the fold nor the enzymatic activity of UBC13 . NMR and kinetics studies indicate that deamidation does not alter the active site of UBC13 . Instead , it reduces the interaction between UBC13 and RING domains to diminish the catalytic effect of the RING . The interaction with TRAF6 is inhibited because E100 in dUBC13 competes with an intermolecular R14/E69 salt-bridge at the dUBC13/TRAF6RING interface to form an intramolecular salt-bridge , which destabilizes the native complex . The new intramolecular salt-bridge also inhibits the long-range transient interactions formed by R14 with TRAF6RING . In addition , repulsive interactions between E100 and the negatively charged interface of TRAF6RING destabilize the transient complexes . Cumulatively , these mechanisms reduced the binding energy between UBC13 and TRAF6RING by 1 . 69 kcal/mol . Consequently , its efficiency in activating the UBC13 ~Ub conjugate drops by 6-fold ( Figure 10F ) . The effect of deamidation is also manifested in another UBC13/RING complex . Deamidation reduced the rate of Ub-discharge and the rate of Ub2 synthesis by several folds in the UBC13/RNF38RING complex ( Figure 10F and N ) . These results provide first insights into the molecular mechanism behind the inactivation of the Ubiquitin pathway by bacterial deamidation . For several E3s like RNF38 , the RING domain can function as a monomer ( Buetow et al . , 2015 ) . For others like TRAF6 , the RING domains function as dimers ( Middleton et al . , 2017; Yin et al . , 2009 ) . Since deamidation perturbed the binding of UBC13 to both TRAF6 and RNF38 , it is likely that deamidation inactivates UBC13 for both homo/hetero-dimeric RING-E3s . In dimeric RINGs like TRAF6 , the proximal RING interacts with one unit of UBC13 ~ Ub , and the distal RING interacts with the donor Ub of another UBC13 ~ Ub . Additionally , for TRAF6 dimer , the first zinc-finger following the RING domain of one protomer also interacts with the donor Ub of the UBC13 ~ Ub bound to another protomer ( Middleton et al . , 2017; Figure 10—figure supplement 3 ) . However , the binding between UBC13 and TRAF6RING is independent of dimerization ( Yin et al . , 2009 ) , indicating that binding between UBC13 and proximal RING is primary . Indeed , deamidation inhibited the binding of UBC13 to both TRAF6RING and to the longer dimeric TRAF6RZ3 , confirming that secondary interactions between Ub/RING or Ub/zinc-finger cannot compensate for the loss of primary E2/RING interaction . Though the interface of a protein complex has numerous interacting residues , a few hotspot residues contribute significantly to the binding energy than the rest ( Moreira et al . , 2007 ) . Hotspot prediction algorithms reported R14 to be a hotspot at the UBC13/TRAF6RING interface ( Meireles et al . , 2010; Zhu and Mitchell , 2011 ) . Analysis of all the UBC13/RING ( or U-box ) structures shows that intermolecular salt-bridges involving R14 are present in several of the UBC13/RING complexes ( Figure 9—figure supplement 1 ) . Binding studies by isothermal titration calorimetry measured that R14A substitution reduced the binding energy of UBC13/ZNRF1RING complex by ~1 . 2 kcal mol−1 ( Behera et al . , 2018 ) . Similarly , our NMR titrations measured that R14A substitution reduced the binding energy by ~1 . 4 kcal mol−1 in the UBC13/TRAF6RING complex . Altogether , R14 appears to be a hotspot at the UBC13/TRAF6RING interface . Deamidation of UBC13 disrupts the salt-bridge between R14 and E69 due to competition from E100 , which destabilizes the UBC13/TRAF6RING complex . Recently , the salt-bridge competition or ‘theft’ mechanism was observed in the binding switch of Raf Kinase Inhibitory Protein ( RKIP ) to either Raf-1 or GPCR-kinase 2 ( Skinner et al . , 2017 ) . Phosphorylation of S153 in RKIP created a new salt-bridge between pS153 and K157 , which disrupted the pre-existing salt-bridges involving K157 and resulted in the local unfolding of RKIP . The unfolding inhibited binding of RKIP to Raf-1 but promoted its binding to GPCR-kinase 2 . Unlike RKIP , deamidation does not alter the local fold of UBC13 . While intramolecular salt-bridges compete within RKIP , the competition is between an intermolecular and intramolecular salt-bridge in UBC13 . Besides the hotspots , electrostatic substitutions at the vicinity of the interface can considerably increase protein-protein association and affinity ( Selzer et al . , 2000 ) . Such substitutions would shift the equilibrium between native and transient complexes ( Volkov et al . , 2010 ) . The proper conformation of transient complexes is vital for two proteins to form a stable , productive complex ( Pan et al . , 2019; Schilder and Ubbink , 2013 ) . How PTMs affect native protein-protein interactions observed in the ground state structure of the complex is well appreciated . However , little is known about how PTMs can affect higher energy transient complexes . PTMs like phosphorylation , eliminylation , and deamidation can change the surface electrostatics of a protein ( Ribet and Cossart , 2010 ) to modulate the conformation and population of transient complexes significantly . By a combination of all-atom simulations and experimental data , this study provides molecular details of how PTMs can modulate transient protein-protein association to inhibit interaction . In the dUBC13/RNF38 complex , deamidation majorly alters transient association . Nonetheless , this causes a 10-fold drop in the catalytic activity of the complex ( Figure 10N ) , indicating that modifying the transient protein-protein association can severely affect function . The fate of the polyubiquitinated substrate depends on the linkage specificity of the conjugated polyubiquitin chain , which in turn depends on the specificity of the E2/E3 interaction . Rationally designed E2/E3 interactions can change the fate and function of cellular proteins ( van Wijk et al . , 2009 ) . Hence , the interfacial contacts that determine E2/E3 specificities are a subject of intense research ( Christensen et al . , 2007; Soss et al . , 2011; van Wijk et al . , 2009; van Wijk et al . , 2012 ) . The strength and functionality of E2/E3 interactions have been typically tested by mutating the interfacial residues ( Christensen et al . , 2007; Das et al . , 2009; Das et al . , 2013 ) . This work provides evidence that transient interactions have an equally significant role in E2/E3 interaction and function . Rational design strategies will improve significantly by incorporating transient E2/E3 interactions . In several cases , the native interactions cannot explain the functional implications of PTMs . This study introduces a new approach to understand the functional role of PTMs by considering their transient interactions . For example , spontaneous deamidation ( non-enzymatic ) is rampant in age-onset diseases , including neurodegenerative and ocular diseases . Deamidation of amylin accelerates its aggregation and amyloid formation ( Dunkelberger et al . , 2012 ) . Crystallins deamidate with age , which promotes its aggregation to trigger cataract ( Pande et al . , 2015 ) . Crystallin surfaces are highly charged , and possibly deamidation alters their charge distribution to promote non-native association leading to aggregation . However , the sites of deamidation on crystallins do not provide a clear mechanism of the process . Deamidation-induced modulation of transient protein-protein association may explain the tendency of these proteins to aggregate . The bacterial effector OspI from S . flexneri presents a fascinating case , wherein a pathogen employs subtle mechanisms like salt-bridge competition and modification of transient protein-protein association , to produce a remarkable cumulative effect of inhibiting protein-protein interaction , polyubiquitination , and the host immune response . Such a mechanism could be a ubiquitous mode of regulating cellular pathways by PTMs .
The interfacial contacts of UBC13/TRAF6RING were obtained from PDB entry 3HCU using the typical cut-offs in the contact analysis tool in UCSF Chimera ( Pettersen et al . , 2004 ) . Initial structures of UBC13 and TRAF6RING for MD simulations and association rate constant ( kon ) estimation were obtained from the PDB entry 3HCU ( Chain A/B ) , which represents a model of the native complex . For MD simulations of TRAF6RING , a C-terminal truncated model comprising of residues 50–148 was used . Substitutions were introduced by replacing existing sidechain with the best aligning rotamer from the Dunbrack rotamer library ( Dunbrack , 2002 ) in UCSF Chimera ( Pettersen et al . , 2004 ) . Electrostatic surface potentials of UBC13 and TRAF6RING were calculated using the Adaptive Poisson-Boltzmann Solver ( Baker et al . , 2001 ) . All simulation methodologies employed in the study were performed using the AMBER99SB-ILDN force field ( Best and Hummer , 2009; Hornak et al . , 2006; Lindorff-Larsen et al . , 2010 ) . Unbiased MD simulations were performed in GROMACS version 4 . 6 . 4 , while biased simulations were performed using the pull code in Gromacs 5 . 1 . 2 ( Abraham et al . , 2015; Hess et al . , 2008 ) . All the acidic and basic residues apart from Histidines were modeled in their charged states . The Zinc AMBER force field ( Peters et al . , 2010 ) parameters were used to model the two zinc coordination sites within the TRAF6RING domain . The initial structures were solvated in an appropriate box using the TIP3P ( Jorgensen et al . , 1983 ) water model . The non-bonded ion parameters proposed by Joung and Cheatham ( 2008 ) were used to model Na+ and Cl- ions in TIP3P water . Additional non-bonded parameter corrections proposed by Yoo and Aksementiev for cation-chloride ( Yoo and Aksimentiev , 2012 ) , amine-carboxylate ( Yoo and Aksimentiev , 2016b ) and aliphatic carbon-carbon ( Yoo and Aksimentiev , 2016a ) interactions were used for all simulations to eliminate overestimation of the strength of these interactions . A suitable number of counterions were added to neutralize the residual charge of the system , and additional ions were added to the box depending on the desired concentration . The electrically neutral , solvated system was then subjected to energy minimization using the steepest descent method for a maximum of 5000 steps until the maximum force on any atom was less than 1000 kJ mol−1 nm−1 . Production simulations were performed under periodic boundary conditions at a temperature of 300 K , and 1 bar pressure ( NPT ensemble ) following equilibration carried out for 600 ps with a two fs time step . Temperature control was achieved using the v-rescaling thermostat ( Bussi et al . , 2007 ) for both equilibration ( Tc = 0 . 1 ps ) and production ( Tc = 2 . 5 ps ) steps . The Berendsen ( Berendsen et al . , 1984 ) ( Tp = 1 ps ) and Parrinello-Rahman ( Parrinello and Rahman , 1981 ) barostat ( Tp = 5 ps ) were employed for pressure control during equilibration and production steps respectively . All bond lengths were constrained using the LINCS ( Hess , 2008 ) algorithm . Virtual interaction sites were employed for hydrogen atoms ( Bjelkmar et al . , 2010 ) , which permitted the use of a five fs time step . Short-range electrostatics and van der Waals interactions were calculated using a 1 . 0 nm cut-off . Long-range electrostatics were calculated using the smooth Particle Mesh Ewald ( PME ) method ( Darden et al . , 1993; Essmann et al . , 1995 ) . An analytical dispersion correction was applied to approximate the effect of long-range van der Waals interactions . Simulations of free dUBC13 and UBC13/dUBC13 native complexes with TRAF6RING were performed using the general MD protocol described above . Salt-bridge occupancies were computed using a cut-off distance of 0 . 5 nm between arginine Cζ and glutamate Cδ atoms using a python script . For unbiased-association simulations , UBC13/dUBC13 and TRAF6RING were separated along with the x component of the vector connecting their center of masses by 4 nm in a cubic box with an edge length of 12 . 6 nm . This results in a box volume equal to ~2000 nm3 and the resulting concentration of each protein species equal to ~1 mM . Fifteen independent association runs ( 100 ns each with different initial atomic velocities ) were performed at 10 mM NaCl and 100 mM NaCl concentrations . Trajectories were analyzed every 250 ps using analysis scripts available within the Gromacs package . Minimum distances were measured between UBC13 and TRAF6RING to identify native-like , non-native , and non-associating trajectories using g_mindist . Native-like transient complex formation was considered to occur in a trajectory when the minimum distance between the sidechain nitrogen atoms of R6/K10/R14 ( UBC13 ) and sidechain oxygen atoms of D57/E69 ( TRAF6 RING ) fell below 0 . 35 nm and persisted for more than eight ns . Among the remaining trajectories , minimum distances were measured between all UBC13 and TRAF6 RING heavy atoms . Non-native and non-associating trajectories were then identified using the same distance and persistence period cut-off . The spatial distribution of TRAF6RING COMs around UBC13/dUBC13 was obtained by calculating the displacement between COMs of Cα atoms of helix-I in UBC13 and TRAF6RING ( 70-109 ) using g_dist and plotted for all fifteen association trajectories . RMSD-based structural clustering ( Daura et al . , 1999 ) with a cut-off of 0 . 45 nm was performed on pairwise rmsd matrices ( determined using g_rmsd ) of the combined trajectories ( 1 . 5 μs ) using UBC13/dUBC13 ( Cα atoms ) as a reference for superposition to obtain the UBC13/TRAF6RING transient complex clusters . The rmsd was computed for the backbone atoms of both UBC13 and TRAF6RING between all trajectory snapshots . The output structures of the complex were then superposed on UBC13 ( PDB: 3HCU ) to obtain the location of TRAF6RING cluster representatives . Native complex formation was identified by measuring the Cα rmsd of TRAF6RING with respect to its position in the crystal complex for trajectories which exhibited the formation of native-like transient complexes using UBC13 ( Cα atoms ) as a reference for superposition . Two-dimensional free energy landscapes for UBC13/dUBC13 association with TRAF6RING were computed as a function of minimum distance ( for salt-bridges between UBC13-R6/K10/R14 and TRAF6-D57/E69 ) and TRAF6RING rmsd using a python script . Bin width of 0 . 02 nm was used for both coordinates , and the free energy ( ΔG ) corresponding to each bin were determined using the relation: ( 2 ) ΔG ( R1 , R2 ) =−KBT[InPi−InPmax]where R1/R2 are the two reaction coordinates , kB is the Boltzmann constant , T is the temperature , Pi is the joint probability of R1/R2 in a given bin and Pmax is the max value of the probability . The lowest free energy state corresponds to ΔG = 0 . Visualization of MD trajectories and analysis of the native contacts was performed using the MD analysis tool in UCSF Chimera . Umbrella sampling is an enhanced-sampling method ( Torrie and Valleau , 1977 ) to determine free energy differences for state transformations by calculating the potential of mean force ( PMF ) as the function of a predefined reaction coordinate ( ξ ) . This amounts to performing multiple , equilibrium MD simulations along with a range of ξ values and determining the corresponding free energy ( A ( ξ ) ) . At points along ξ which are referred to as windows , a harmonic restraining potential ( wi ) is added to the original Hamiltonian in order to restrain the system close to a target value ( ξi ) : ( 3 ) Eb ( r ) =Eu ( r ) +wi ( ξ ) ( 4 ) wi ( ξ ) =k/2∗ ( ξ−ξi ) 2 In Equation ( 2 ) , E ( r ) denotes the potential energy function ( force field ) of the system as a function of atomic coordinates ( r ) . Subscripts b and u indicate biased and unbiased energies . k is the force constant for the harmonic bias potential whose magnitude determines the range of ξ values sampled within each window . The application of the bias potential particularly improves sampling in high energy regions along ξ which are poorly sampled in conventional MD simulations . The modified energy function yields biased probability distributions ( Pib ( ξ ) ) of ξ from which the unbiased , free energy ( Ai ( ξ ) ) of a window can be computed using the relation: ( 5 ) Ai ( ξ ) =−kBTlnPib ( ξ ) −wi ( ξ ) +Fi The value of Fi needs to be determined for all windows to combine adjacent windows and obtain the global free energy profile ( A ( ξ ) ) , which describes state transformation . The weighted histogram analysis method ( WHAM ) ( Kumar et al . , 1992 ) is widely used to determine optimal values of Fi in an iterative fashion so as to obtain convergence of the free energy profile . Similar to previous studies which utilized the US approach to study the energetics of protein-protein association , the center of mass ( COM ) separation along the x-axis was chosen as the reaction coordinate to calculate the PMF for Barnase/Barstar and UBC13/TRAF6 RING association . Barstar and UBC13 were translated along the x-axis of the vector joining the COM between their binding partners to positions separated by 0 . 1 nm intervals to achieve final separations of 4 . 0 and 4 . 5 nm respectively . Each window was simulated in a rectangular box of dimensions 12 . 6 nm x 9 . 0 nm x 9 . 0 mm following equilibration . During production MD ( 10 ns for each window ) , a harmonic restraining potential ( k = 1500 kJ mol−1 nm−2 ) was applied to the COM vector in each window and data were collected every 250 fs . The biased COM probability distributions from each window were analyzed based on WHAM and combined to obtain the global free energy ( PMF ) profile using the g_wham analysis tool ( Hub et al . , 2010 ) . A total of 200 bins were used to construct a mean PMF profile from five independent time blocks ( 1 . 5 ns each from 2 . 5 to 10 ns ) for each window ( Supplementary file 1-table S2 ) . A tolerance of 10−9 was used to check for convergence . The free energy of association ( ΔGPMF ) along the COM coordinate was determined by setting the free energy profile to zero in the unbound state ( 4 . 0 nm for Barnase/Barstar and 4 . 5 nm for UBC13/TRAF6RING ) and determining the value of the profile at mean COM separation in the native complex ( 2 . 3 nm for Barnase/Barstar and 2 . 7 nm for UBC13/TRAF6RING ) window during production MD . Coulombic interaction energies from the US windows were calculated using g_energy tool in Gromacs . Steered MD is a non-equilibrium , enhanced-sampling technique wherein a time-dependent biasing potential is applied on a selected atom/COM of atomic groups to induce perturbation in the system ( Grubmüller et al . , 1996 ) . This is achieved by means of a moving dummy atom attached to the pull group via a stiff harmonic spring . This method has been previously employed to study the unfolding of protein domains and the dissociation of protein-ligand complexes . In this study , we carried out force-induced unbinding of TRAF6RING from UBC13 using the constant velocity pulling method along the x-axis to a COM separation of ~ 6 nm in 30 ns long simulations . The external force ( Fext ) acting on the pull group as the simulation progresses with time ( t ) is defined by the relation: ( 6 ) Fext=k ( vt−x ) where k is the spring constant , v is the pull velocity , and x is the displacement of the ligand from its initial position . The pull force was applied to COM of Cα atoms of the RING domain ( residues 70–109 ) at a low pull rate of 0 . 125 nm ns−1 ( k = 1500 kJ mol−1 nm−2 ) . The same box dimensions were used as in the case of umbrella sampling . The COM of Cα atoms of UBC13 was kept fixed by periodically removing its rotational and translation motion to promote dissociation of the complex . The cumulative work ( W ) done to separate complex was calculated using the relation: ( 7 ) W=∫F . dx=1/2∑ ( fi+fi ) ( Xi+1−Xi ) The work done over two successive simulation steps ( i and i+1 ) is calculated as a product of the average force ( f ) multiplied by the displacement of the ligand between the steps . W is thus calculated by numerically integrating the work done between all successive steps of the simulation . The force and COM position of TRAF6RING was recorded every five ps . Fmax ( rupture force ) is defined as the maximum value of the force recorded in the Force-COM extension profile of an individual or averaged SMD trajectory . W was defined at a COM separation of 4 . 5 nm in the Work-COM extension profile as all native contacts were found to be disrupted at this COM separation . Both Fmax and W correlate with the binding affinity of the complex and hence allow for an analysis of the effect of mutations on the stability of the complex . The association rate constants ( kon ) for UBC13/dUBC13/R14A-UBC13 with TRAF6RING were calculated based on transient complex theory ( Alsallaq and Zhou , 2008 ) . The crystallographic complexes were provided as inputs to the TransComp webserver ( Qin et al . , 2011 ) . The server computes the association rate constant ( kon ) from a basal association rate constant ( ka0 ) in the absence of electrostatic interactions and the electrostatic free energy ( ΔGel ) of a pre-generated transient complex ensemble using the relation: ( 8 ) kon=kaoexp ( −ΔGel/kT ) where k and T are the Boltzmann constant and temperature , respectively . The kon values for all complexes were computed at ionic strengths of 10 and 100 mM NaCl . To generate the transient complex ensemble from the crystallographic complex , proteins are treated as rigid bodies and configurations are randomly sampled along six relevant degrees of freedom . Three degrees of freedom are for relative rotation and three for relative translation . Sampling is performed such that the distance between the center of the binding sites of the interacting proteins remains within a suitable cutoff ( rcut ) . The desired value of rcut is determined in an iterative fashion by varying from 0 . 6 to 1 . 0 nm ( increment of 0 . 1 nm ) so as to generate 107 clash-free configurations . The number of intermolecular contacts ( both native and non-native ) is calculated for all configurations . The standard deviation in the rotation angle ( σχ ) is calculated for configurations at each contact level ( Nc ) . The transient complex ensemble corresponds to the value of Nc at which the difference between σχ ( Nc ) and the average of all lower contact levels reaches a maximum . From the transient complex ensemble , the electrostatic interaction energy ( Uel ) was calculated for each configuration as ( 9 ) Uel=Uel ( AB ) −Uel ( A ) −Uel ( B ) where Uel ( AB ) corresponds to the interaction energy of the complex and Uel ( A/B ) corresponds to that of the two unbound proteins . Electrostatic energies are calculated using the linearized Poisson-Boltzmann equation . ΔGel is calculated as a simple average over interaction energies of the transient complex ensemble . ka0 is determined from 4000 force-free Brownian dynamics trajectories based on a previously developed algorithm ( Zhou , 1993 ) . All videos were prepared by pre-processing trajectories using a lowpass filter ( g_filter tool in Gromacs ) followed by the recording of trajectory snapshots in UCSF Chimera using the MD analysis module . In all trajectories , rotational and translational motion of UBC13 was removed entirely to fix its position with respect to TRAF6RING . A synthetic construct for TRAF6RING ( residues 50–124 ) was synthesized at Thermo Fischer Scientific , USA . PCR amplified ORF was cloned into a pGEX4T1 vector using NEB HF cloning kit ( New England Biolabs , Inc USA ) . E69A-TRAF6 was cloned using PCR based site-directed mutagenesis . Variants of UBC13 were generated using PCR based site-directed mutagenesis in WT-UBC13 in pET24 vector ( a gift from Prof . Rachel Klevit ) . RNF38 plasmid was a gift from Prof . Danny Huang . TRAF6RZ3 plasmid was a gift from Prof . Catherine Day . All the plasmids were confirmed by sequencing . All proteins were expressed in BL21 DE3 star cells ( Invitrogen ) , grown at 37°C ( in LB media for unlabeled proteins and in M9 media for C-13 or N-15 labeled proteins ) till OD600 reached 0 . 7 and were induced with 0 . 5 mM IPTG for 4–5 hr . The harvested cells were lysed in 50 mM Tris , 250 mM NaCl , 5 mM βME , 2% glycerol , 0 . 01% triton-X , 1 mM PMSF and DNase at pH 7 . 5 using Emulsiflex high-pressure homogenizer . GST-TRAF6RING and GST-RNF38RING ( residues 387–465 ) were bound to GSTrap HP columns ( GE Healthcare ) and were eluted with 10 mM reduced glutathione . The proteins were further purified by gel filtration on Superdex 75 pg 16/600 ( GE Healthcare ) in 25 mM Tris , 100 mM NaCl and 5 mM βME at pH 7 . 5 . The His-tagged UBC13 and its variants were bound to HisTrap HP columns ( GE Healthcare ) and were eluted with 100 mM-500mM Imidazole gradient . The proteins were further purified by gel filtration on Superdex 75 pg 16/600 ( GE Healthcare ) in 25 mM Tris , 100 mM NaCl and 5 mM βME at pH 7 . 5 . TRAF6RZ3 was induced at OD600 ~ 0 . 7 with 0 . 2 mM IPTG and 0 . 1 mM ZnCl2 . The lysed cells were resuspended in 50 mM Tris , 350 mM NaCl , 7 mM βME , 1 mM PMSF , 10 mg Lysozyme , 0 . 01% Triton X , pH 8 and centrifuged . The supernatant was bound to HisTrap beads and was with 150 mM-450mM Imidazole gradient . The eluent was subsequently purified by gel filtration on Superdex 75 pg 16/600 ( GE Healthcare ) in 25 mM Tris , 100 mM NaCl and 5 mM βME at pH 7 . 5 . For the purification of Ub and its variants , lysed cells were resuspended in lysis buffer ( 50 mM Sodium acetate , 5 mM BME , 0 . 01% Triton X , pH 4 . 5 ) , lysed by sonication and centrifuged to pellet cell debris . The supernatant was passed through the SP FF column ( GE Healthcare ) and the protein was eluted by gradient elution with increasing salt concentration ( 0 mM NaCl- 600 mM NaCl ) . The proteins were further purified by gel filtration chromatography on Superdex 75 16/600 column ( GE Healthcare ) in 50 mM Sodium Phosphate , 100 mM NaCl , 5 mM BME , pH 6 . 5 . For the purification of control K63-linked di-Ub , 1 µM UbE1 , 40 µM Ubc13 , 40 µM GB1-MMS2 , 40 µM GST-RNF38 , 200 µM D77-Ub and 200 µM K63A-Ub were mixed with reaction buffer ( 50 mM Sodium Phosphate , 5 mM MgCl2 , 3 mM ATP , 0 . 5 mM DTT , pH 6 . 5 ) overnight . The mixture was dialyzed in 50 mM Sodium Acetate , 5 mM BME , pH 4 . 5 , and centrifuged . The supernatant was passed through the SP FF column ( GE Healthcare ) and the di-Ub was separated by gradient elution with increasing salt concentration ( 0 mM NaCl- 600 mM NaCl ) . All NMR titration experiments were recorded at 298K on 600 MHz or 800 MHz Bruker Avance III HD spectrometer with a cryoprobe head . The samples were prepared in 25 mM Tris , 100 mM NaCl , pH 7 . 5 , and 10% D2O . For NMR titration experiments , either ~ 2 mM TRAF6RINGor ~ 1 mM RNF38RING were titrated into ~ 0 . 15 mM 15N-UBC13 , 15N-dUBC13 and other mutants . The titration data was fit in 1:1 protein:ligand model using the equation CSPobs = CSPmax { ( [P]t+[L]t+Kd ) - [ ( [P]t+[L]t+Kd ) 2- 4[P]t[L]t]1/2}/2[P]t , where [P]t and [L]t are total concentrations of protein and ligand at any titration point . The Arginine sidechain spectra of UBC13 and dUBC13 were recorded at 298K on 800 MHz Bruker Avance III HD spectrometer with a cryoprobe head , processed with NMRpipe ( Delaglio et al . , 1995 ) and analyzed with Sparky ( Kneller and Kuntz , 1993 ) . The samples were prepared in 25 mM phosphate , 50 mM NaCl , pH 6 . 0 . D2O was not directly added to the NMR sample to avoid additional signals due to 2H isotopomers . For NMR lock , the 3 mm NMR tube was inserted co-axially in an external tube containing D2O . The arginine sidechain Hε , Nε , Cζ resonances were assigned by broadband NOESY-HSQC , HNCACB ( Junji Iwahara and Marius Clore , JBNMR 2006 ) , 2D HD ( CD ) NE and HD ( CDNE ) CZ ( Frans Mulder JACS 2007 ) experiments . The solution structure of UBC13/RNF38RING complex was calculated in HADDOCK ( Dominguez et al . , 2003 ) using the structure of UBC13 ( PDB ID: 3HCT ) and RNF38RING ( PDB ID: 4V3K ) . Rigid body energy minimization generated one thousand initial complex structures , and the 200 lowest energy structures were selected for torsion angle dynamics and subsequent Cartesian dynamics in an explicit water solvent . Default scaling for energy terms was applied . The interface of proteins was kept semi-flexible during simulated annealing and the water refinement steps . The statistics of the highest scored ensemble are , HADDOCK score: −80 . 6 ± 0 . 5 , Cluster size: 74 , RMSD from the overall lowest-energy structure: 0 . 8 ± 0 . 5 , Van der Waals energy: −44 . 4 ± 4 . 4 , Electrostatic energy: −169 . 9 ± 19 . 6 , Desolvation energy: −8 . 6 ± 5 . 1 and Buried Surface Area: 1274 . 1 ± 75 . 3 . For ubiquitination assay using TRAF6RZ3 , E1 ( 0 . 5 μM ) , UBC13 and MMS2 ( 20 μM ) and Alexa Fluor Maleimide ( Invitrogen ) labeled UbS20C ( 20 μM ) were incubated with TRAF6RZ3 ( 15 μM ) in UB-buffer ( 50 mM Tris , 5 mM ATP , 5 mM MgCl2 , 20 mM NaCl , 2 mM DTT , pH 8 ) at 37°C for 10 min . The same reaction was repeated with GST-TRAF6RING instead of TRAF6RZ3 . The reaction mixtures were separated on 12% SDS gel , and the images were acquired in Uvitec ( Cambridge ) . For ubiquitination assay using GST-RNF38RING , E1 ( 0 . 5 μM ) , UBC13 and MMS2 ( 5 μM ) and Alexa Fluor Maleimide ( Invitrogen ) labeled UbS20C ( 20 μM ) were incubated with GST-RNF38RING ( 3 μM ) in 20 mM Tris , 5 mM ATP , 5 mM MgCl2 ( pH 7 . 5 ) at 37°C for 30 min . The reaction mixtures were separated in 12%–15% SDS gel , and the images were acquired in iBright FL1000 ( Invitrogen ) . For Ub conjugation assays , E1 ( 1 μM ) , UBC13/dUBC13 ( 20 μM ) , and K63A-Ub ( 100 μM ) were incubated in UB-buffer at 37°C for 15 min . For discharge assays , E1 ( 1 μM ) , UBC13/dUBC13 ( 20 μM ) , and K63A-Ub ( 100 μM ) were incubated in UB-buffer at 37°C for 60 min . The reaction was quenched with EDTA ( 30 mM ) at room temperature for 5 min . MBP-Mms2 ( 40 μM ) , TRAF6RING or RNF38RING ( 40 μM ) and Lysine ( 15 mM ) were added to start Ub-discharge . The reaction was quenched by 4X non-reducing SDS loading buffer at desired time-points . For Ub2 synthesis kinetics , E1 ( 1 μM ) , UBC13/dUBC13 ( 20 μM ) , and K63A-Ub ( 100 μM ) were incubated in UB-buffer at 37°C for 60 min . The reaction was quenched with EDTA ( 30 mM ) at room temperature for 5 min . Then MBP-Mms2 ( 40 μM ) , RNF38RING ( 40 μM ) and D77-Ub ( 20–100 μM ) were added to start Ub2 synthesis . The reaction was quenched by 4X non-reducing SDS loading buffer at desired time-points . The reaction mixtures of Ub conjugation , Ub discharge , and Ub2 synthesis were separated in 12% SDS gel , stained overnight with SYPRO Ruby ( Invitrogen ) and the images were acquired in Uvitec ( Cambridge ) . For the pull-down of TRAF6RZ3 with UBC13 ~ Ub conjugate , Ub conjugation reaction was performed as described above , and the reaction was quenched with EDTA at room temperature for 5 min . MBP-Mms2 ( 80 μM ) was incubated on amylose bead ( NEB ) in 50 mM Tris , 20 mM NaCl , pH 8 . The UBC13 ~ Ub conjugate ( 20 μM ) was then mixed with TRAF6RZ3 ( 60 μM ) and MBP-Mms2 on the amylose beads for 1 hr at 4°C , washed ( 3x , each time centrifuged at 3500 rpm for 3 min ) . After the final wash , the pellet was resuspended in 50 mM Tris , 20 mM NaCl , pH 8 and separated on 12% SDS gel . For the pull-down of UBC13 ~ Ub conjugate with GST-RNF38RING , the GST-RNF38RING was incubated with glutathione agarose ( Thermo Fisher ) . The UBC13 ~ Ub conjugate was mixed with GST-RNF38RING ( 60 μM ) on glutathione agarose for 1 hr at 4°C , washed ( 3x ) , resuspended in 50 mM Tris , 20 mM NaCl , pH 8 and separated on 12% SDS gel . The gels for both pull-down assays were stained overnight with SYPRO Ruby ( Invitrogen ) , and the images were acquired in Uvitec ( Cambridge ) .
The deamidase OspI from enteric bacteria Shigella flexneri deamidates a glutamine residue in the host ubiquitin-conjugating enzyme UBC13 and converts it to glutamate ( Q100E ) . Consequently , its polyubiquitination activity in complex with the RING-finger ubiquitin ligase TRAF6 and the downstream NF-κB inflammatory response is silenced . The precise role of deamidation in silencing the UBC13/TRAF6 complex is unknown . We report that deamidation inhibits the interaction between UBC13 and TRAF6 RING-domain ( TRAF6RING ) by perturbing both the native and transient interactions . Deamidation creates a new intramolecular salt-bridge in UBC13 that competes with a critical intermolecular salt-bridge at the native UBC13/TRAF6RING interface . Moreover , the salt-bridge competition prevents transient interactions necessary to form a typical UBC13/RING complex . Repulsion between E100 and the negatively charged surface of RING also prevents transient interactions in the UBC13/RING complex . Our findings highlight a mechanism wherein a post-translational modification perturbs the conformation and stability of transient complexes to inhibit protein-protein association .
Shigella is a highly infectious group of bacteria that attack the human digestive tract , causing severe and often deadly diarrhoea , especially in children . There is currently no vaccine to protect against the disease , and some strains are also now resistant to antibiotics . People get infected by eating or drinking contaminated foods and water . After passing through the stomach , Shigella invades and then multiplies in the lining of the intestine , eventually causing tissue damage and irritation . During this process , Shigella ‘hides’ from its host’s immune system by blocking how intestinal cells respond to infection . Normally , infected cells send out chemical signals that act like a call for help , attracting specialised immune cells to clear the infection . In intestinal cells , two proteins called UBC13 and TRAF6 work together to switch on this response . Specifically , TRAF6 needs to bind to UBC13 for the switch to turn on . Like many proteins , UBC13 is formed of thousands of atoms; some of these are organized in ‘functional groups’ , a collection of atoms joined in a specific manner and with special chemical properties . During Shigella infection , the bacteria produce an enzyme that changes a single functional group ( an amino group ) at a specific location within UBC13 for a different one ( an hydroxyl group ) . Previous research showed that this could stop the immune response in intestinal cells , but the mechanism remained unknown . Mohanty et al . therefore set out to determine exactly how a change of so few atoms could have such a dramatic effect . Biochemical studies using purified proteins revealed that Shigella’s alteration to UBC13 did not change its overall structure . However , the altered protein could no longer bind to its partner TRAF6 . Theoretical analysis and computer simulations revealed that the normal binding process relies on a positively charged amino acid ( one of the protein’s building blocks ) in UBC13 and a negatively charged one in TRAF6 being attracted to each other . Shigella’s substitution , however , introduces a second negatively charged amino acid in UBC13 . This ‘steals’ the positively charged amino acid that would normally interact with TRAF6: the electrical attraction between the two proteins is disrupted , and this stops them from binding . The work by Mohanty et al . reveals the exact mechanism Shigella uses to dampen its host’s immune response during infection . In the future , this knowledge could be used to develop more effective drugs that would help control outbreaks of diarrhoea .