basement membranes are specialized extracellular matrices that underlie epithelial cell sheets and surround muscle , fat and peripheral nerve cells .
basement membranes are crucially involved in many developmental processes and their integrity is essential for tissue function in adult animals .
the main basement membrane proteins are among the most highly conserved extracellular proteins ; they include laminins , type iv collagen , nidogen and perlecan .
basement membrane assembly begins with the polymerization of laminin into a cell - associated network ( yurchenco et al , 2004 ) .
mutations of tissue - specific laminin genes cause severe diseases in humans , such as congenital muscular dystrophy and epidermolysis bullosa ( miner & yurchenco , 2004 ) .
laminins are large , cross - shaped molecules consisting of three polypeptide chains (  ,  and  ) .
the three short arms of the cross are composed of one chain each , whereas the long arm is a coil containing all three chains that terminates in a tandem of five linkage group domains provided by the -chain ( fig 1a ) .
the human genome encodes five - , three - and three -chains which are assembled into at least 15 laminin heterotrimers ( aumailley et al , 2005 ) . with the exception of the 3a , 4 and 2 chains ,
all short arms have a single laminin amino - terminal ( ln ) domain at their distal end , followed by a tandem of laminin - type epidermal growth factor - like ( le ) domains .
the same domain architecture is also found in netrins , an important class of axon guidance molecules ( fig 1a ; dickson , 2002 ) .
laminin-111 ( 111 ) polymerization in vitro requires calcium and a full complement of ln domains , suggesting that the nodes in the polygonal laminin-111 network contain one 1 , one 1 and one 1 ln domain ( three - arm interaction model ; yurchenco et al , 1985 ; paulsson , 1988 ; yurchenco & cheng , 1993 ; mckee et al , 2007 ) . in support of this model ,
mutations in the ln domains of the 1 and 2 chains compromise laminin function in vivo ( xu et al , 1994 ; patton et al , 2008 ; edwards et al , 2010 ) .
binding experiments with short - arm fragments showed heterotypic interactions that were consistent with the three - arm interaction model (  ,  ,  ) , as well as unexpected homotypic  interactions ( odenthal et al , 2004 ) .
a molecular understanding of laminin polymerization has been hindered by the lack of structural information about the ln domain . in this study
we report the crystal structure and functional analysis of a laminin 5ln  lea12 fragment .
as ln domains can not be produced in isolation ( ettner et al , 1998 ; odenthal et al , 2004 ) , we produced a series of laminin short - arm fragments containing the ln domain , followed by 24 lea domains ( supplementary table s1 online ) .
the best crystals we obtained were of an 5ln  lea12 fragment and diffracted to approximately 3.4  resolution .
individual mutations of the four predicted n - linked glycosylation sites ( n100a , n148 t , n248e , n383e ) showed that glycosylation of asn 148 and asn 248 was required for protein solubility .
lea12 n100a / n383e double mutant diffracted to 2.9  resolution and was used to determine the structure . the laminin 5ln 
lea12 structure resembles a flower , with the ln and le domains forming the head and stalk , respectively ( fig 1b , c ) .
the head measures approximately 40  40  25  and the stalk is approximately 60  long . when the head is viewed from its narrow side , it appears tilted by approximately 25 relative to the straight stem .
the le domains of 5ln  lea12 have the canonical le fold , that was previously established by the crystal structure of laminin 1leb24 ( stetefeld et al , 1996 ) .
the 5lea12 domains almost entirely consist of irregular coils and are stabilized by a continuous ladder of disulphide bridges ( fig 1b ) .
the eight canonical cysteines in each domain are linked 13 , 24 , 56 and 78 , creating four loops , which we refer to as the 23 , 34 , 56 and 78 loops . in 5lea1 , there is a fifth disulphide bridge linking the 56 and 78 loops .
the long 34 loops of each le domain contain short antiparallel -strands and protrude similarly to leaves from the stalk .
the ln domain is a richly elaborated antiparallel -sandwich , with strands 18 forming a jelly - roll motif ( fig 1b , c ) . a dali search for related structures ( holm & rosenstrm , 2010 ) showed that the core of the ln domain is most similar to bacterial galactose binding domains ( gbds ; supplementary fig s1a online ) .
this relationship was recognized in a recent modelling study ( kalkhof et al , 2010 ) .
however , the ln fold has several additions to the jelly - roll motif that have no counterpart in gbds .
the two -sheets of the jelly - roll motif in 5ln are extended by two short strands , 1 and 5. the latter strand is part of a larger insertion between 5 and 6 that also contains a conserved disulphide bridge between cys 200 and cys 220 ( fig 1d ) .
additional large insertions between 6 , 7 and 8 contain several -helices that contribute substantially to the bulk of the ln domain .
residues 70119another insertion into the jelly - roll motif  are disordered in the 5ln 
the disordered region contains four conserved cysteines , but otherwise its sequence is highly variable in the ln domain superfamily ( fig 1d ) .
there is no obvious linker segment between the ln and lea1 domains ( fig 1b , c ) .
the two domains interact through a large , partly disjointed , interface that buries a total of approximately 2,500  of solvent - accessible surface area .
half of the buried area is accounted for by the interaction of an extended n - terminal segment with lea1 .
most notably , the side chain of pro 52 is accommodated in a pocket between the 13 and 24 disulphide bridges of lea1 ( supplementary fig s2 online ) .
pro 52 is strictly conserved in all other ln domains ( fig 1d ) , suggesting that the ln 
the other contacts between the ln and lea1 domains of laminin 5 involve the long 34 and 78 loops of lea1 and are not as well conserved across the ln superfamily . the 5ln 
lea1 interface which interact with the side chains of his 57 , his 158 , arg 256 , lys 260 , arg 304 and his 308 ( supplementary fig s2 online ) .
arg 256 and lys 260 are involved in proteoglycan - mediated cell adhesion to the 5 short arm ( nielsen & yamada , 2001 ) .
the phosphate ions might thus act similarly to the sulphate groups of heparan sulphate chains .
bacterial gbds  which the core of the ln fold is most closely related to 
contain a structural calcium binding site at the carboxy terminus of helix 1 ( boraston et al , 2004 ) .
lea12 structure , even though suitable acidic ligands are present in the 5 chain ( asp 127 and asp 298 ; supplementary fig s1b , c online ) .
to exclude the possibility that calcium was lost from 5ln  lea12 during crystallization at ph 45 , we tested for calcium binding at neutral ph by measuring the intrinsic fluorescence of 5ln  lea12 ( the putative calcium binding site is close to two fluorophores , trp 132 and trp 133 ) .
the fluorescence spectra in the presence and absence of calcium were identical ( supplementary fig s1d online ) , indicating that the n - terminal region of the 5 chain does not bind to calcium .
this conclusion agrees with the results of circular dichroism spectroscopy experiments ( odenthal et al , 2004 ) .
the sites responsible for the calcium dependence of laminin polymerization ( yurchenco et al , 1985 ; paulsson , 1988 ) remain unknown . having determined the first structure of an ln fragment , we sought to identify the ln domain regions responsible for laminin - network formation .
we thought that residues mediating network interactions might be conserved in all -chains , as laminin heterotrimers with different -chains can form mixed polymers ( cheng et al , 1997 ) .
mapping the conserved residues of mouse laminin -chains onto the 5ln domain structure showed that the 638 face of the -sandwich is more highly conserved than the opposite face ( not shown ) . remarkably , two conserved surface patches remain even when invertebrate laminin -chains are included in the analysis ( fig 2a , supplementary fig s3 online ) .
patch 1 is situated on the 12745 face and comprises glu 178 , pro 189 , arg 265 and arg 267 .
however , the glycan attached to asn 148 blocks access to these residues in 5ln .
all ln domains contain a predicted glycosylation site in this region , either at the start of 2 as in 5ln or at the end of 1 ( fig 1d , supplementary fig s3 online ) .
patch 2 is on the 638 face and comprises trp 132 and asn 168 , as well as pro 229 , leu 230 and glu 231 in the 56 loop ( fig 2b ) .
importantly , there are no glycosylation sites obstructing patch 2 in any laminin -chain ( fig 2a ) .
further , with the exception of trp 132 , patch 2 residues are not conserved in laminin - and -chains ( fig 1d ) .
these observations do not support a structural role for patch 2 and instead imply that it has a specific role in laminin -chain function . to test whether patch 2 is important for 5ln  lea12 function , we introduced three mutations into the 56 loop : l230a , e231k and e234k .
the first two residues are strictly conserved in all -chains , whereas glu 234 is conserved in all -chains except in drosophila wing blister ( supplementary fig s3 online ) . as a control
, we mutated a surface - exposed glutamic acid on the opposite face of the ln domain ( e202k ) .
all mutants were secreted as efficiently as wild - type protein by hek 293 cells , indicating correct folding . to determine the effects of the mutations , we used an established functional assay in which laminin-111 polymerization is inhibited by soluble short - arm fragments ( yurchenco & cheng , 1993 ; cheng et al , 1997 ; garbe et al , 2002 ) .
lea12 protein robustly inhibited laminin-111 polymerization , so that most of the laminin-111 protein remained in the soluble fraction ( fig 2c , d ) .
the e202k control mutant was a similarly potent inhibitor . by contrast , all three 5ln  lea12 proteins with mutations in the 56 loop did not inhibit laminin-111 polymerization , demonstrating that the 56 loop of laminin 5ln interacts with a crucial network - forming site on laminin-111 . as 56 loop residues are strictly conserved in all laminin -chains of invertebrates and vertebrates ( fig 2a ) ,
it is likely that this ln region is involved in the network interactions of other laminins .
binding experiments with recombinant ln  lea14 fragments of the 1 , 5 , 1 and 1 chains detected all possible heterotypic interactions , as well as homotypic 11 and 55 interactions ( odenthal et al , 2004 )
. however , this binding pattern is inconsistent with results from an earlier study using proteolytic laminin-111 fragments , which found only a single interaction of measurable affinity ( binary 11 or ternary 111 ; yurchenco & cheng , 1993 ) .
we used surface plasmon resonance ( spr ) binding experiments to study the interactions between the ln 
we detected a weak interaction ( estimated kd of 5 m ) between 1 and 1 ( fig 3b , c ) , but , in contrast to the results of odenthal et al ( 2004 ) , 5 did not bind to 1 , 1 or to itself in our experiments ( fig 3d , e and data not shown ) .
most interestingly , when we flowed a 1:1 mixture of 5 and 1 over a 1 surface , we observed a larger response than for 1 alone , indicative of a ternary 511 complex ( fig 3d ) .
the same ternary complex could also be assembled on a 1 surface ( fig 3e ) .
the ternary 511 interaction showed slower kinetics than the binary 11 interaction , possibly indicating that 5 binds to a 11 heterodimer .
although the underlying equilibria are probably more complex , the association and dissociation phases of the ternary complex ( fig 3d ) could be fitted ( =1.9 ) by a 1:1 langmuir model with a kd of 0.8 m .
the 5 l230a mutant  which was inactive in the polymerization assay ( fig 2c , d)also formed a ternary complex with 1 and 1 , but the mutant complex dissociated substantially faster than the wild - type complex ( fig 3d ) .
thus , mutations in the 56 loop of 5ln seem to destabilize the ternary 511 complex .
as the dissociation of the mutant complex could not be described by a single rate constant , it was not possible to quantify the destabilizing effect . in conclusion , the results of our spr binding studies with
laminin-511 short - arm fragments support the original three - arm interaction model of laminin-111 polymerization ( yurchenco & cheng , 1993 ; mckee et al , 2007 ) .
we propose that transient contacts between the - and -short arms are consolidated by the slower incorporation of an -short arm , and that the conserved 56 loop of ln domains has an important function in the cooperative assembly of the ternary  nodes in the laminin network .
as ln domains can not be produced in isolation ( ettner et al , 1998 ; odenthal et al , 2004 ) , we produced a series of laminin short - arm fragments containing the ln domain , followed by 24 lea domains ( supplementary table s1 online ) .
the best crystals we obtained were of an 5ln  lea12 fragment and diffracted to approximately 3.4  resolution .
individual mutations of the four predicted n - linked glycosylation sites ( n100a , n148 t , n248e , n383e ) showed that glycosylation of asn 148 and asn 248 was required for protein solubility .
lea12 n100a / n383e double mutant diffracted to 2.9  resolution and was used to determine the structure .
lea12 structure resembles a flower , with the ln and le domains forming the head and stalk , respectively ( fig 1b , c ) .
the head measures approximately 40  40  25  and the stalk is approximately 60  long . when the head is viewed from its narrow side , it appears tilted by approximately 25 relative to the straight stem .
the le domains of 5ln  lea12 have the canonical le fold , that was previously established by the crystal structure of laminin 1leb24 ( stetefeld et al , 1996 ) . the 5lea12 domains almost entirely consist of irregular coils and are stabilized by a continuous ladder of disulphide bridges ( fig 1b ) .
the eight canonical cysteines in each domain are linked 13 , 24 , 56 and 78 , creating four loops , which we refer to as the 23 , 34 , 56 and 78 loops . in 5lea1
the long 34 loops of each le domain contain short antiparallel -strands and protrude similarly to leaves from the stalk .
the ln domain is a richly elaborated antiparallel -sandwich , with strands 18 forming a jelly - roll motif ( fig 1b , c ) . a dali search for related structures ( holm & rosenstrm , 2010 ) showed that the core of the ln domain is most similar to bacterial galactose binding domains ( gbds ; supplementary fig s1a online ) .
this relationship was recognized in a recent modelling study ( kalkhof et al , 2010 ) .
however , the ln fold has several additions to the jelly - roll motif that have no counterpart in gbds .
the two -sheets of the jelly - roll motif in 5ln are extended by two short strands , 1 and 5. the latter strand is part of a larger insertion between 5 and 6 that also contains a conserved disulphide bridge between cys 200 and cys 220 ( fig 1d ) .
additional large insertions between 6 , 7 and 8 contain several -helices that contribute substantially to the bulk of the ln domain .
the disordered region contains four conserved cysteines , but otherwise its sequence is highly variable in the ln domain superfamily ( fig 1d ) .
there is no obvious linker segment between the ln and lea1 domains ( fig 1b , c ) .
the two domains interact through a large , partly disjointed , interface that buries a total of approximately 2,500  of solvent - accessible surface area .
half of the buried area is accounted for by the interaction of an extended n - terminal segment with lea1 .
most notably , the side chain of pro 52 is accommodated in a pocket between the 13 and 24 disulphide bridges of lea1 ( supplementary fig s2 online ) .
pro 52 is strictly conserved in all other ln domains ( fig 1d ) , suggesting that the ln 
the other contacts between the ln and lea1 domains of laminin 5 involve the long 34 and 78 loops of lea1 and are not as well conserved across the ln superfamily . the 5ln 
lea1 interface which interact with the side chains of his 57 , his 158 , arg 256 , lys 260 , arg 304 and his 308 ( supplementary fig s2 online ) . arg 256 and lys 260 are involved in proteoglycan - mediated cell adhesion to the 5 short arm ( nielsen & yamada , 2001 ) .
the phosphate ions might thus act similarly to the sulphate groups of heparan sulphate chains .
bacterial gbds  which the core of the ln fold is most closely related to 
contain a structural calcium binding site at the carboxy terminus of helix 1 ( boraston et al , 2004 ) .
lea12 structure , even though suitable acidic ligands are present in the 5 chain ( asp 127 and asp 298 ; supplementary fig s1b , c online ) .
to exclude the possibility that calcium was lost from 5ln  lea12 during crystallization at ph 45 , we tested for calcium binding at neutral ph by measuring the intrinsic fluorescence of 5ln
 lea12 ( the putative calcium binding site is close to two fluorophores , trp 132 and trp 133 ) .
the fluorescence spectra in the presence and absence of calcium were identical ( supplementary fig s1d online ) , indicating that the n - terminal region of the 5 chain does not bind to calcium .
this conclusion agrees with the results of circular dichroism spectroscopy experiments ( odenthal et al , 2004 ) .
the sites responsible for the calcium dependence of laminin polymerization ( yurchenco et al , 1985 ; paulsson , 1988 ) remain unknown .
having determined the first structure of an ln fragment , we sought to identify the ln domain regions responsible for laminin - network formation .
we thought that residues mediating network interactions might be conserved in all -chains , as laminin heterotrimers with different -chains can form mixed polymers ( cheng et al , 1997 ) .
mapping the conserved residues of mouse laminin -chains onto the 5ln domain structure showed that the 638 face of the -sandwich is more highly conserved than the opposite face ( not shown ) . remarkably , two conserved surface patches remain even when invertebrate laminin -chains are included in the analysis ( fig 2a , supplementary fig s3 online ) .
patch 1 is situated on the 12745 face and comprises glu 178 , pro 189 , arg 265 and arg 267 .
however , the glycan attached to asn 148 blocks access to these residues in 5ln .
all ln domains contain a predicted glycosylation site in this region , either at the start of 2 as in 5ln or at the end of 1 ( fig 1d , supplementary fig s3 online ) .
patch 2 is on the 638 face and comprises trp 132 and asn 168 , as well as pro 229 , leu 230 and glu 231 in the 56 loop ( fig 2b ) .
importantly , there are no glycosylation sites obstructing patch 2 in any laminin -chain ( fig 2a ) .
further , with the exception of trp 132 , patch 2 residues are not conserved in laminin - and -chains ( fig 1d ) . these observations do not support a structural role for patch 2 and instead imply that it has a specific role in laminin -chain function . to test whether patch 2 is important for 5ln  lea12 function , we introduced three mutations into the 56 loop : l230a , e231k and e234k .
the first two residues are strictly conserved in all -chains , whereas glu 234 is conserved in all -chains except in drosophila wing blister ( supplementary fig s3 online ) . as a control , we mutated a surface - exposed glutamic acid on the opposite face of the ln domain ( e202k ) .
all mutants were secreted as efficiently as wild - type protein by hek 293 cells , indicating correct folding . to determine the effects of the mutations , we used an established functional assay in which laminin-111 polymerization is inhibited by soluble short - arm fragments ( yurchenco & cheng , 1993 ; cheng et al , 1997 ; garbe et al , 2002 ) .
lea12 protein robustly inhibited laminin-111 polymerization , so that most of the laminin-111 protein remained in the soluble fraction ( fig 2c , d ) .
by contrast , all three 5ln  lea12 proteins with mutations in the 56 loop did not inhibit laminin-111 polymerization , demonstrating that the 56 loop of laminin 5ln interacts with a crucial network - forming site on laminin-111 .
as 56 loop residues are strictly conserved in all laminin -chains of invertebrates and vertebrates ( fig 2a ) , it is likely that this ln region is involved in the network interactions of other laminins .
lea14 fragments of the 1 , 5 , 1 and 1 chains detected all possible heterotypic interactions , as well as homotypic 11 and 55 interactions ( odenthal et al , 2004 ) .
however , this binding pattern is inconsistent with results from an earlier study using proteolytic laminin-111 fragments , which found only a single interaction of measurable affinity ( binary 11 or ternary 111 ; yurchenco & cheng , 1993 ) .
we used surface plasmon resonance ( spr ) binding experiments to study the interactions between the ln 
we detected a weak interaction ( estimated kd of 5 m ) between 1 and 1 ( fig 3b , c ) , but , in contrast to the results of odenthal et al ( 2004 ) , 5 did not bind to 1 , 1 or to itself in our experiments ( fig 3d , e and data not shown ) .
most interestingly , when we flowed a 1:1 mixture of 5 and 1 over a 1 surface , we observed a larger response than for 1 alone , indicative of a ternary 511 complex ( fig 3d ) . the same ternary complex could also be assembled on a 1 surface ( fig 3e ) .
the ternary 511 interaction showed slower kinetics than the binary 11 interaction , possibly indicating that 5 binds to a 11 heterodimer .
although the underlying equilibria are probably more complex , the association and dissociation phases of the ternary complex ( fig 3d ) could be fitted ( =1.9 ) by a 1:1 langmuir model with a kd of 0.8 m .
the 5 l230a mutant  which was inactive in the polymerization assay ( fig 2c , d)also formed a ternary complex with 1 and 1 , but the mutant complex dissociated substantially faster than the wild - type complex ( fig 3d ) .
thus , mutations in the 56 loop of 5ln seem to destabilize the ternary 511 complex .
as the dissociation of the mutant complex could not be described by a single rate constant , it was not possible to quantify the destabilizing effect . in conclusion
, the results of our spr binding studies with laminin-511 short - arm fragments support the original three - arm interaction model of laminin-111 polymerization ( yurchenco & cheng , 1993 ; mckee et al , 2007 ) .
we propose that transient contacts between the - and -short arms are consolidated by the slower incorporation of an -short arm , and that the conserved 56 loop of ln domains has an important function in the cooperative assembly of the ternary  nodes in the laminin network .
complementary dnas coding for ln  lea12 and ln  lea14 fragments of the mouse laminin 1 , 2 , 5 , 1 and 1 chains ( supplementary table s1 online ) were obtained by pcr amplification from plasmids kindly provided by takako sasaki ( university of erlangen , germany ) .
the pcr products were cloned into a modified pcep - pu vector coding for proteins with a c - terminal his6-tag ( carafoli et al , 2009 ) .
proteins were purified from the conditioned medium of episomally transfected hek293 c18 cells ( carafoli et al , 2009 ) by histrap affinity chromatography ( ge healthcare ) , followed by size exclusion chromatography on a s200 hr10/30 column ( ge healthcare ) at 4c using a running buffer of 20 mm na - hepes , ph 7.5 , 150 mm nacl , 2 mm cacl2 . crystal structure determination .
crystals of fully glycosylated laminin 5ln  lea12 were obtained by 200 nl sitting drop vapour diffusion using a protein concentration of 8 mg / ml and 4% peg6000 , 0.1 m mgcl2 , 0.1 m sodium acetate , ph 5.2 as precipitant .
heavy atom derivatives were prepared by soaking crystals for 18 h in 2 mm uo2(no3)2 or 1 mm k2ptcl4 .
crystals were flash - frozen in liquid nitrogen in mother liquor supplemented with 20% glycerol .
diffraction data were collected at the diamond light source ( native data , beamline io2 , =0.980  ) and at the swiss light source ( derivative data , beamline pxiii , =1.000  ) .
the diffraction limit was anisotropic ( approximately 3.2  in the ab plane and approximately 4  along c ) .
the images were processed with imosflm ( www.mrc-lmb.cam.ac.uk/harry/imosflm ) and with programs of the ccp4 suite ( ccp4 , 1994 ) .
a miras - phased electron density map at 3.4  resolution was obtained with autosharp ( vonrhein et al , 2007 ) .
a partial model was built with o ( jones et al , 1991 ) and refined with cns ( brnger et al , 1998 ) .
the model was completed using a native data set to 2.9  resolution ( approximately 3.2  along c ) collected from an isomorphous crystal of the laminin 5ln 
lea12 n100a / n383e double mutant obtained at 6 mg / ml protein concentration and 0.4 m nh4h2po4 ( ph 4.0 ) .
briefly , mouse laminin-111 ( sigma ) at a concentration of 0.25 mg / ml ( 0.35 m ) was incubated with 1 mm cacl2 or 10 mm edta in 50 mm tris 
hcl , ph 7.4 , 150 mm nacl , 0.1% triton x-100 for 3 h at 37c in a total volume of 100 l .
the pellet and supernatant fractions were analysed by sds  polyacrylamide gel electrophoresis and coomassie blue staining .
band intensities ( 1 and 1/1 combined ) were quantified with imagej ( rsbweb.nih.gov/ij ) .
the spr experiments were performed at 25c using a biacore 3000 instrument ( ge healthcare ) .
proteins were coupled to cm5 chips in 10 mm sodium acetate , ph 4.5 , according to the manufacturer 's instructions .
the running buffer was 20 mm na - hepes , ph 7.5 , 150 mm nacl , 2 mm cacl2 at a flow rate of 30 l / min .
 determinants of laminin polymerization revealed by the structure of the 5 chain amino - terminal regionlaminin polymers are a key component of all basement membranes . 
 the first crystal structure of a laminin amino - terminal domain identifies a conserved surface loop in laminin alpha chains that is involved in polymer formation . 
Introduction
Results and Discussion
Crystallization of a laminin short-arm fragment
Laminin 5LNLEa12 structure
A motif involved in laminin-network formation
Methods
Supplementary Material