Exploring the inhibition mechanism of adenylyl cyclase type 5 by n-terminal myristoylated Gαi1
Many proteins are involved in cell communication of which one type is the G-protein-coupled receptor ( GPCR ) , embedded in the membrane ., GPCRs are part of a major signalling pathway , the GPCR signal transduction pathway , which enables the transfer of a signal from the extracellular region to the intracellular side and is a key target for drug development ., A large diversity of GPCRs can be found in nature as about 800 human genes are involved in storing different types of GPCRs that can interact with neurotransmitters , hormones or exogenous ligands , for example 1 ., In the cytosol , G proteins , composed of an α , β and γ subunit , are the first interaction partner of activated GPCRs ., When a heterotrimeric G protein is activated by a GPCR , the trimer dissociates , resulting in an α subunit and a βγ dimer 2 ., Activated Gα subunits transport the signal from the membrane to other regions of the cell by stimulating or inhibiting reactions via protein-protein interactions ., Besides direct activation by GPCRs , the function of G proteins can also be influenced by other environmental factors , such as lipidation ., Permanent N-myristoylation , for instance , is known to change the structure and function of the inhibitory G-protein subunit Gαi1 in its active GTP-bound state 3–6 ., While a wide range of GPCRs exists , a relatively low diversity is present in the G protein family , e . g . in the human body ., The human body includes only a relatively small variety of 21 α , 6 β and 12 γ subunits 1 ., The Gα subunits are divided into four major subfamilies based on their sequence homology and function 7: stimulatory Gαs , inhibitory Gαi , Gαq and Gα12 8 , 9 ., Overall the structures of the Gα subfamilies are similar ( S1 Fig , Fig 1 ) , including a Ras domain and an alpha helical ( AH ) domain ., The Ras domain is present in all members of the G-protein superfamily and can perform hydrolysis of GTP to GDP during deactivation of the Gα subunit 10 ., In addition , the domain includes an interaction site for GPCRs as well as regions that can interact with the βγ dimer ., Moreover , the Ras domain can also undergo lipidation ., Except for Gαt , all Gα proteins are able to reversibly bind a palmitate to their N-terminal helix ., Besides palmitoylation , Gαi can also permanently bind a myristoyl moiety to the N-terminus that appears to be crucial for the function of the subunit ( Fig 1C ) 4 , 5 , 9 , 11 ., The AH domain is unique to the Gα subfamilies , which is composed of six α helices and interacts with the Ras domain when GTP or GDP is present ( Fig 1C ) ., However , this interaction between the AH and Ras domain is weakened when a nucleotide is absent in Gα’s active site 12–14 ., The high structural similarity among members of the Gα subfamilies is illustrated by aligning the X-ray structures of stimulatory Gαs and inhibitory Gαi1 , resulting in a root mean square deviation ( RMSD ) of only 1 . 07 Å between the Cα atoms of the two structures ( Fig 1A ) 15 , 16 ., Hence , from a comparison of the structures it is difficult to conclude what the origin is of their inverse action , i . e . , how the structure can be related to a stimulatory respectively an inhibitory effect ., An example of a protein in which both Gαi and Gαs are important for regulation is adenylyl cyclase ( AC ) ., Ten isoforms of AC are known of which nine are membrane-bound ( AC1-9 ) and one is soluble ( sAC ) 17 ., These different types of AC are found throughout the body in different concentrations ., AC5 , for instance , is present in high quantities in the brain , the spinal cord and the heart , and is associated to congestive heart failure and pain perception 18 , 19 ., G proteins have the ability to either stimulate ( Gs ) or inhibit ( Gi ) adenylyl cyclases’ conversion of adenosine triphosphate ( ATP ) to cyclic adenosine monophosphate ( cAMP ) and pyrophosphate 20 , 21 ., ACs consist of two membrane-bound regions , each built from six trans-membrane domains , and a catalytic region in the cytosol that includes two pseudo-symmetric domains , C1 and C2 ( Fig 1 ) 22 ., GTP-bound Gαs is known to bind to the C2 domain for which the interaction site is known from X-ray structures of Gαs interacting with AC ( Fig 1B ) 23 ., Such data is absent for the case of Gαi ., In the absence of direct experimental information , a putative interaction site of GTP-bound Gαi has been suggested in analogy to the known structure of the complex of Gαs and AC ( Gαs:AC ) as the pseudo-symmetric site on the C1 domain ( Fig 1B ) ., However , how the interaction of Gαi on the C1 domain should induce inhibition is not obvious 5 ., Furthermore , since with this hypothesis the interaction sites of Gαi1 and Gαs are highly similar in addition to their structures , it is unclear how the α subunits can differentiate the two binding sites on AC and what the cause is of the stimulatory versus the inhibitory effect induced by the subunits ., A factor that could play an important role in differentiating the action of Gαi and Gαs is the difference in lipidation of both subunits ., Although the X-ray structures in the Protein Data Base ( PDB ) 24 of the active inhibitory and stimulatory Gα subunits tightly align , the N-terminus , which is not resolved for Gαi or Gαs , is not myristoylated during the expression process of Gαi as lipidation can hinder crystallisation 4 ., Hence , it is not clear to what extent the missing N-terminal myristoyl moiety affects the Gαi structure of the remaining protein while the bound myristoyl group has been known to be crucial for Gαi’s conformation and function as the ability to interact with AC5 is abolished upon removal of the myristate 4–6 ., Classical molecular dynamics ( MD ) simulations of myristoylated GTP-bound Gαi1 , Gαi1myr , demonstrate the stability of the myristoyl moiety on the Ras domain due to a hydrophobic pocket formed by β2-β3 , α1 and the C-terminus α5 ( Fig 1C ) and show that myristoylation can have a significant effect on the conformation of the subunit 25 ., The findings suggest the possibility of an alternative novel interaction mode and open up new possibilities for selective interactions with AC ., This is because the found structural changes in the classical MD simulations of Gαi1myr 25 suggest that the subunit will not be able to interact with C1 as Gαs interacts with C2 ., Here , we investigate the interaction between Gαi1myr and AC , using classical MD simulations ., To this end , the initial structure of Gαi1myr was taken from reference 25 in which a 2 μs classical MD simulation of Gαi1myr is described ., Gαi1myr can inhibit only particular isoforms of AC: AC1 , AC5 , AC6 26 ., In this study AC5 is used because X-ray structures of AC’s catalytic domains are composed of isoforms AC2 and AC5 ., Ca ., 16 AC structures can be found in the PDB with different resolutions and/or crystallisation conditions ., All available structures have been co-crystallised with a Gαs subunit and correspond therefore to stimulated conformations at various levels of activation , depending on the nature of bound cofactors ( e . g . cofactor-free complex of AC , substrate-bound AC complex ) ., When AC5 becomes active , roughly three conformational options are possible: a complex of Gαs and AC5 , Gαs:AC5 , Gαs in complex with ATP-bound AC5 , Gαs:AC5 ( ATP ) , or a complex of Gαs and AC5 bound to the reaction products cAMP and pyrophosphate , Gαs:AC5 ( cAMP ) ., Currently , it is not known which one of these forms is most likely to interact with Gαi1myr , or if Gαi1myr can inhibit all of them ., In this study , the structure of the AC5 protein was taken from a crystal structure of the cofactor-free Gαs:AC5 complex ., This apo AC5 structure was used as it could provide insight into Gαi1myr’s inhibitory effect on a stimulated conformation of AC5 in the absence of ATP ., The selected AC5 structure was employed to build a Gαi1myr:AC5 complex ( Fig 1C ) and to explore if the binding of Gαi1myr is able to affect the active conformation initially induced by Gαs ., The absence of ATP in the active site provides the opportunity to investigate Gαi1myr’s ability to prevent the formation of AC’s fully activated form by altering AC’s conformation unfavourably prior to substrate association ., In order to verify which changes are due to the interaction of AC5 with Gαi1myr and which alterations are a result of the removal of Gαs , a second simulation of AC5 , with the Gαs subunit removed , was performed on the same time scale as the Gαi1myr:AC5 complex ., Hence , in this study the impact of the presence of myristoylated Gαi on the function of AC5 is explored via investigating the conformational features of the Gαi1myr:AC5 and the free AC5 complex ( a system that only includes AC’s catalytic region in solution ) in comparison with the Gαs:AC X-ray structure ., The Gαi1myr:AC5 complex has been obtained via docking the Gαi1myr structure on to the C1 domain of AC5 ., Already the initial docking results confirm the possible importance of the myristoyl-induced structural changes of Gαi1myr as a new interaction mode for Gαi1myr could be identified ., The comparison of the performed classical MD simulation ( 2 . 5 μs ) of the Gαi1myr:AC5 complex and the free AC5 system suggest two possible ways of AC inhibition in its apo form ., First , Gαi1myr seems to inhibit AC’s conversion of ATP to cAMP by preventing active-site formation as the Gαi1myr subunit perturbs the conformation of the active site at the C1/C2 interface ., Second , the effect of Gαi1myr on the AC structure leads to a closed conformation of the Gαs binding site on C2 , decreasing the probability of Gαs association and thus of a counter-balancing re-stimulation of the AC5 activity ., Taken together , the observed events lead to a suggestion for a putative Gαi1myr inhibition mechanism of apo AC5 in which lipidation is crucial for Gαi1myr’s function and its protein-protein interactions ., Hence , the results of this study provide a possible indication that lipidation could play a significant role in regulating G protein function and therefore could impact signal transduction in G protein mediated pathways 4–6 ., The PDB structure 1AZS , including the Gαs:AC complex with AC in the apo form , was used as a template , including 1AZS’s C1 and C2 domain , for the initial AC5 structure of Rattus norvegicus ( UniprotKB Q04400 ) 27–29 ., The structure of the Rattus norvegicus Gαi1myr subunit ( UniprotKB P10824 ) interacting with GTP and Mg2+ was taken from reference 25 ( S2 and S3 Figs ) ., The HADDOCK web server 30 was used for docking ten conformations of Gαi1myr on the catalytic domains of AC5 of Rattus norvegicus ., The Gαi1myr snapshots were extracted at the end of the Gαi1myr classical MD trajectory ( around 1 . 9 μs ) discussed in reference 25 , with a time interval of 0 . 5 ns ., The active region of Gαi1 was defined in HADDOCK as a large part of the AH domain ( 112-167 ) , the switch I region ( 175-189 ) and the switch II region ( 200-220 ) , allowing for a large unbiased area on the Gαi1myr protein surface to be taken into account during docking ., The active region of AC5’s C1 domain was defined as the α1 helix ( 479-490 ) and the C-terminal region of the α3 helix ( 554-561 ) because experimentally it has been found that Gαi1myr is unable to interact with C2 and its main interactions with AC are with the C1 domain 5 ., Passive residues , residues that could take part in protein-protein interaction , were defined as residues around the active residues that are on the protein surface and within a radius of 6 . 5 Å of any active residue 30 ., The initial Gαi1myr:AC5 complex for the classical MD simulations was selected based on ( 1 ) the absence of overlap between the C2 domain and Gαi1myr , ( 2 ) no overlap with Gαi1myr’s GTP binding region and the interaction site of Gαi1myr with C1 and ( 3 ) presence of similar complexes in the top-ten docking results of the docking calculations performed for all ten Gαi1myr snapshots ., The first property of the selection criteria is important since Gαi1myr is unable to interact with C2 5 ., The second criterium has been defined since GTP is located in the active site of Gαi1myr in the classical MD simulations , but was not incorporated in the docking procedure because this is not possible in HADDOCK ., Therefore , no overlap between the GTP binding site and the C1 domain should be present in the docking result as otherwise the GTP molecule will not be able to fit in Gαi1myr’s active site ., The last criterium is the presence of similar Gαi1myr:AC5 complexes of the selected complex in all top-ten docking results which increases the probability that complexation of the two proteins is not conformation specific , but is robust as similar complexes can be obtained using different conformations of Gαi1myr ., The Gαi1myr:AC5 complex was used to simulate the protein complex for 2 . 5 μs at 310 K and 1 bar using a Nosé-Hoover thermostat and an isotropic Parrinello-Rahman barostat ., In the active site of Gαi1myr one Mg2+ ion and a GTP molecule are present ., In order to closer mimic an AC5 system with which ATP or a product such as pyrophosphate would be able to interact , a Mg2+ ion was added to the active site of AC5 ( see S1 Appendix ) ., Additionally , about 68 000 water molecules and 150 mM KCl are present in the simulated system ., The force fields used for the protein and the water molecules are AMBER99SB 31 and TIP3P 32 , which were employed by Gromacs 4 . 6 . 6 33 , 34 to perform the runs ., For GTP , the force field generated by Meagher et al . was used 35 ., The adjusted force field parameters for the K+ ions and the Cl- ions were taken from Joung et al . 36 ., The Mg2+ ion parameters originated from Allnér et al . 37 and the parameter set for the myristoyl group was taken from reference 25 ., The charges for the myristoyl group were obtained with Gaussian 09 38 based on Hartree Fock calculations in combination with a 6-31G* basis set and using the AMBER RESP procedure 39 ., Appropriate atom types from the AMBER99SB force field were selected to complete the myristoyl description ., Electrostatic interactions were calculated with the Ewald particle mesh method with a real space cutoff of 12 Å ., Bonds involving hydrogen atoms were constrained using the LINCS algorithm 40 ., The time integration step was set to 2 fs ., The free AC5 system was simulated with the same setup as the Gαi1myr:AC5 complex ., The system was solvated in 30 000 water molecules and a 150 mM KCl concentration ., The initial location of the Mg2+ ion in the active site of the enzyme was the same as in the Gαi1myr:AC5 complex system ., Multiprot 41 and VMD 42 were used to align protein structures ., Uniprot 43 was used to align protein sequences ., Images were prepared with VMD 42 ., The initial conformation of the Gαi1myr:AC5 complex suggests that Gαi1myr’s proposed interaction site ( see S1 Appendix ) affects the conformation of C1 in a different way than Gαs stabilises the C2 domain ( Figs 1 , 2 and S3 Fig ) ., Unlike Gαs , Gαi1myr is not located between the helices of AC5’s catalytic domain , but appears to clamp the C1 domain into its inactive conformation ., Gαi1myr is positioned around AC5’s α3 , interacting with α1 , α2 , and α3 via its switch I , II and III region together with the C-terminal domain of αB ( Fig 2 and S3 Fig ) ., Since C1’s α1 helix appears to decrease its distance with respect to the C2 domain when an ATP analog , adenosine 5- ( α-thio ) -triphosphate ( ATPαS ) , is present in the active site ( S2 Fig ) , the interactions between Gαi1myr and C1’s α1 in the Gαi1myr:AC5 complex could suggest that one way by which Gαi1myr is able to inhibit ATP’s conversion is by preventing C1’s α1 to rearrange upon ATP binding ., The simulation of Gαi1myr:AC5 in comparison with the free AC5 trajectory and the Gαs:AC X-ray structure demonstrate that the first step in decreasing AC5’s activity in the apo form is the relocation of the β7-β8 loop ( Fig 7 , step one ) ., In fact , the β7-β8 loop seems to have an important role for the stimulatory response since the presence of Gαs leads to the stabilisation of the loop , forming ATP’s binding site ( Fig 7 , starting conformation of AC in left panel ) 23 ., This loop conformation is lost as soon as Gαs is absent , as observed for both free AC5 and Gαi1myr:AC5 ., In step two of Fig 7 the Gαi1myr:AC5 complex undergoes a rearrangement in the C2 domain ( absent in free AC5 ) , which leads to a further perturbation of AC5’s active site ., The classical molecular dynamics simulations also show that in the presence of Gαi1myr , there appears to be a decrease in probability for Gαs association ( Fig 7 , step 3 and Fig 6 ) ., Hence , through these rearrangements Gαi1myr could deactivate apo AC5 as well as decrease the probability of reactivation via Gαs ., The results of this study suggest that Gαi1myr deactivates the apo form of adenylyl cyclase type 5 via constraining C1’s active site region ., Inhibition and stimulation of AC5 appear to follow different pathways ., While Gαs binds between the helices of C2 , increasing the stability of the C1:C2 dimer , Gαi1myr is able to clamp the helices of the C1 domain , promoting an inactive conformation of AC5’s catalytic domains and a possible decrease in affinity for Gαs on the C2 domain ., Structurally , Gαs and non-myristoylated Gαi1 are very similar , however , when myristoylation has taken place on the N-terminus of Gαi1 , the conformation of the subunit changes drastically , leading to a structure that differentiates itself from the active Gαs subunit and enables the protein to function in an inhibitory fashion as is shown via the presented classical MD simulations ., Hence , in line with experimental studies , myristoylation appears to be crucial for Gi’s function and demonstrates how important even relatively small changes to a protein structure can be for its function .
Adenylyl cyclase ( AC ) is an important messenger involved in G-protein-coupled-receptor signal transduction pathways , which is a well-known target for drug development ., AC is regulated by activated stimulatory ( Gαs ) and inhibitory ( Gαi ) G proteins in the cytosol ., Although experimental studies have shown that these Gα subunits can stimulate or inhibit AC’s function in a non-competitive way , it is not well understood what the difference is in their mode of action as both Gα subunits appear structurally very similar in a non-lipidated state ., However , a significant difference between Gαs and Gαi is that while Gαs does not require any lipidation in order to stimulate AC , N-terminal myristoylation is crucial for Gαi’s inhibitory function as AC is not inhibited by non-myristoylated Gαi ., At present , only the conformation of the complex including Gαs and AC has been resolved via X-ray crystallography ., Therefore , understanding the interaction between Gαi and AC is important as it will provide more insight into the unknown mechanism of AC regulation ., This study demonstrates via classical molecular dynamics simulations that the myristoylated Gαi1 structure is able to interact with apo adenylyl cyclase type 5 in a way that causes inhibition of the catalytic function of the enzyme , suggesting that Gα lipidation could play a crucial role in AC regulation and in regulating G protein function by affecting Gαi’s active conformation .
Communication between cells is essential for the survival of any multicellular organism ., When these mechanisms cannot function properly , diseases can occur such as heart failure or Parkinson’s disease ., Understanding cell communication is therefore crucial for drug development ., Important proteins in cellular signalling are the ones that initiate mechanisms in the cell after the signal of an extracellular trigger is transported from outside to inside the cell ., G proteins ( GPs ) are an example of such proteins ., Experimental studies have shown that GPs can perform stimulatory or inhibitory functions , however , it is not well understood what the difference is in their mode of action , especially as they are structurally very similar ., Adenylyl cyclase ( AC ) is an enzyme which can be stimulated or inhibited by GPs , depending on which type of GP is active ., Hence , AC is a good candidate for investigating the difference in function between GPs ., However , only the structure of the stimulatory GP interacting with AC is known ., Here , we investigate for the first time the effect of the interaction of an inhibitory GP with AC via classical molecular dynamics simulations in order to obtain a better understanding of the difference between stimulatory and inhibitory GP association and AC regulation .
medicine and health sciences, molecular dynamics, diagnostic radiology, crystal structure, enzymes, condensed matter physics, enzymology, protein structure, lyases, crystallography, g protein coupled receptors, research and analysis methods, solid state physics, imaging techniques, proteins, chemistry, transmembrane receptors, molecular biology, physics, protein structure comparison, biochemistry, signal transduction, biochemical simulations, radiology and imaging, diagnostic medicine, cell biology, adenylyl cyclase, genitourinary imaging, biology and life sciences, physical sciences, computational chemistry, computational biology, macromolecular structure analysis