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Talin is a large cytoskeletal protein that is involved in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton, colocalising with the integrins in focal adhesions (FAs). However, at the leading edge of motile cells, talin colocalises with the hyaluronan receptor layilin in what are thought to be transient adhesions, some of which subsequently mature into more stable FAs. During this maturation process, layilin is replaced with integrins, which are highly clustered in FAs, where localised production of PI(4,5)P(2) by type 1 phosphatidyl inositol phosphate kinase type 1gamma (PIPK1gamma) is thought to play a role in FA assembly. The talin FERM F3 subdomain binds both the integrin beta-subunit cytoplasmic domain and PIPK1gamma, and these interactions are understood in detail at the atomic level. The talin F3 domain also binds to short sequences in the layilin cytoplasmic domain, and here we report the structure of the talin/layilin complex, which shows that talin binds integrins, PIPK1gamma and layilin in similar although subtly different ways. Based on structure comparisons, we designed a set of talin F3 mutations that selectively affected the affinity of talin for its targets, as determined by stopped-flow fluorescence measurements. Such mutations will help to assess the importance of the interactions between talin and its various ligands in cell adhesion and migration.

The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded beta-sandwich fold of the gC1q domain is reduced to nine beta strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the alpha4beta1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of alpha4beta1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu(933)). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-alpha4beta1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site.

Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large "head" on two "legs," with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allowcells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor. © 2011 Cold Spring Harbor Laboratory Press.

Integrins are essential adhesion receptors found on the surfaces of all metazoan cells. As regulators of cell migration and extracellular matrix assembly, these membrane-spanning heterodimers are critical for embryonic development, tissue repair and immune responses. Signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, but integrin affinity for extracellular ligands can also be controlled by intracellular cues. This bidirectional signaling is mediated by the short cytoplasmic tails of the two integrin subunits. Recent structural and functional studies of various integrin fragments and complexes between the cytoplasmic tails and intracellular proteins, such as talin, have provided new insight into the signaling processes centered around the tails, particularly inside-out integrin activation.

Fibronectin, an ubiquitous extracellular matrix (ECM) glycoprotein, plays a major role in fundamental biological processes such as cell adhesion and migration, maintenance of normal cell morphology, cytoskeletal organization, and cell differentiation. Fibronectin is constructed from three types of independently folding protein module (Fn1, Fn2, and Fn3) and is found as a Fibrillar network in the ECM where it interacts with other ECM components and provides anchorage sites for cell surface integrin receptors. The mosaic nature of fibronectin permits it to be analyzed by a "dissection" strategy, where protein fragments generated by recombinant expression in E. coli, P. pastoris, and human cell lines are employed in structural and functional investigations. We describe methods suitable for the production of various fibronectin fragments for study by a variety of techniques including crystallography and electron microscopy but special mention is made of methods suitable for the production of samples for NMR studies.

Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2–F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes. Electronic supplementary material The online version of this article (doi:10.1007/s00232-016-9908-z) contains supplementary material, which is available to authorized users.

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

The phosphorylation state of SpoIIAA is a key factor in the regulation of sporulation in Bacillus subtilis. Previous crystallographic studies had led to the conclusion that phosphorylation alters the binding affinity of SpoIIAA for its partner proteins solely through the additional charge and bulk of the phosphoryl group: small structural changes observed elsewhere in the protein were considered to be random fluctuations rather than the result of phosphorylation. The results presented in the present paper show that NMR studies detect the same subtle structural changes in solution as those seen in the crystal, strongly implying that they are the direct result of phosphorylation. These subtle structural changes are similar to those that occur in a non-phosphorylated mutant that is defective in binding to one of its partner proteins. We propose that the structural changes which occur in SpoIIAA on phosphorylation act in concert with the phosphoryl group to alter its binding properties.

Recent studies of some proteins involved in the formation of focal adhesions are described. These include fibronectin, integrins, talin, Dok1 and filamin. Emphasis is placed on features that facilitate regulated assembly of complexes; these include a modular construction and flexible regions that provide interaction sites whose affinity can be adjusted by conformational masking and phosphorylation. © 2008 Biochemical Society.

Background: Kindlins are essential co-activators of integrins. Results: Kindlin-3 has an elongated structure and forms a ternary complex with the Talin head and integrin β-tails. The Kindlin-3-tail interface involves a membrane-distal NPXY motif on the tail. Conclusion: New information about the conformation and interactions of Kindlin-3 has been obtained. Significance: The solution structure and protein/protein interactions of Kindlin-3 give insight into its role., Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β1A and β1D isoforms, although the interaction is stronger with β1A. An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3.

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16–21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16–24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.

Summary Integrins are cell surface receptors crucial for cell migration and adhesion. They are activated by interactions of the talin head domain with the membrane surface and the integrin β cytoplasmic tail. Here, we use coarse-grained molecular dynamic simulations and nuclear magnetic resonance spectroscopy to elucidate the membrane-binding surfaces of the talin head (F2-F3) domain. In particular, we show that mutations in the four basic residues (K258E, K274E, R276E, and K280E) in the F2 binding surface reduce the affinity of the F2-F3 for the membrane and modify its orientation relative to the bilayer. Our results highlight the key role of anionic lipids in talin/membrane interactions. Simulation of the F2-F3 in complex with the α/β transmembrane dimer reveals information for its orientation relative to the membrane. Our studies suggest that the perturbed orientation of talin relative to the membrane in the F2 mutant would be expected to in turn perturb talin/integrin interactions., Graphical Abstract Highlights ► Simulations and NMR define the interactions of the talin head domain with membranes ► Talin binds to anionic lipids in a bilayer via the F2 subdomain ► Mutations in the F2 lipid-binding site may perturb talin/integrin interactions

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, (6)FnI(1-2)FnII(7-9)FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains (8-9)FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains (6)FnI(1-2)FnII(7)FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains (2)FnII and (7)FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the (8-9)FnI·collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside-out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the beta integrin subunit. Here, we report the first structure of talin bound to an authentic full-length beta integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane-proximal helix of the beta tail disrupts an integrin alpha/beta salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside-out TM signalling.

The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions.

Integrins play a fundamental role in cell migration and adhesion; knowledge of how they are regulated and controlled is vital for understanding these processes. Recent work showed that Dok1 negatively regulates integrin activation, presumably by competition with talin. To understand how this occurs, we used NMR spectroscopy and x-ray crystallography to investigate the molecular details of interactions with integrins. The binding affinities of β3 integrin tails for the Dok1 and talin phosphotyrosine binding domains were quantified using 15N-1H hetero-nuclear single quantum correlation titrations, revealing that the unphosphorylated integrin tail binds more strongly to talin than Dok1. Chemical shift mapping showed that unlike talin, Dok1 exclusively interacts with the canonical NPXY motif of the β3 integrin tail. Upon phosphorylation of Tyr747 in the β3 integrin tail, however, Dok1 then binds much more strongly than talin. Thus, we show that phosphorylation of Tyr747 provides a switch for integrin ligand binding. This switch may represent an in vivo mechanism for control of integrin receptor activation. These results have implications for the control of integrin signaling by proteins containing phosphotyrosine binding domains.

Latrophilin-1 (Lat-1), a target receptor for alpha-Latrotoxin, is a putative G protein-coupled receptor implicated in synaptic function. The extracellular portion of Lat-1 contains a rhamnose binding lectin (RBL)-like domain of unknown structure. RBL domains, first isolated from the eggs of marine species, are also found in the ectodomains of other metazoan transmembrane proteins, including a recently discovered coreceptor of the neuronal axon guidance molecule SLT-1/Slit. Here, we describe a structure of this domain from the mouse Lat-1. RBL adopts a unique alpha/beta fold with long structured loops important for monosaccharide recognition, as shown in the structure of a complex with L-rhamnose. Sequence alignments and mutagenesis show that residues important for carbohydrate binding are often absent in other receptor-attached examples of RBL, including the SLT-1/Slit coreceptor. We postulate that this domain class facilitates direct protein-protein interactions in many transmembrane receptors.

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin beta-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.

Sporulation in Bacillus subtilis serves as a model for the development of two different cell types from a single cell. Although much information has been accumulated about the mechanisms that initiate the developmental programmes, important questions remain that can be answered only by quantitative analysis. Here we develop, with the help of existing and new experimental results, a mathematical model that reproduces published in vitro experiments and explains how the activation of the key transcription factor is regulated. The model identifies the difference in volume between the two cell types as the primary trigger for determining cell fate. It shows that this effect depends on the allosteric behaviour of a key protein kinase and on a low rate of dephosphorylation by the corresponding phosphatase; both predicted effects are confirmed experimentally.

Staphylococcus aureus possesses cell-wall attached proteins that bind the human protein fibronectin (Fn). An intermodule interface between the 4F1 and 5F1 modules in the N-terminal domain of Fn is maintained on bacterial peptide binding but there is a small change in the intermodule orientation and alignment of β-strands that are predicted to bind the peptide. The module pair is elongated, as in the unbound state. Combined with evidence that residues in both 4F1 and 5F1 are directly involved in peptide binding, this observation supports the hypothesis that, when bound to intact Fn, the bacterial protein adopts an unusual, highly extended conformation.

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-π stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein·HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [13C, 15N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.

Focal adhesions (FAs) are large submembrane signaling complexes formed at sites of cellular attachment to the extracellular matrix. The interaction of LD motifs with their targets plays an important role in the assembly of FAs. We have determined the molecular basis for the recognition of two paxillin LD motifs by the FA targeting (FAT) domain of FA kinase using a combination of X-ray crystallography, solution NMR, and homology modeling. The four-helix FAT domain displays two LD binding sites on opposite sites of the molecule that bind LD peptides in a helical conformation. Threading studies suggest that the LD-interacting domain of p95PKL shares a common four-helical core with the FAT domain and the tail of vinculin, defining a structural family of LD motif binding modules.

Activation (affinity regulation) of integrin adhesion receptors controls cell migration and extracellular matrix assembly. Talin connects integrins with actin filaments and influences integrin affinity by binding to the integrins' short cytoplasmic beta-tail. The principal beta-tail binding site in talin is a FERM domain, comprised of three subdomains (F1, F2, and F3). Previous studies of integrin alphaIIbbeta3 have shown that both F2 and F3 bind the beta3 tail, but only F3, or the F2-F3 domain pair, induces activation. Here, talin-induced perturbations of beta3 NMR resonances were examined to explore integrin activation mechanisms. F3 and F2-F3, but not F2, distinctly perturbed the membrane-proximal region of the beta3 tail. All domains also perturbed more distal regions of the beta3 tail that appear to form the major interaction surface, since the beta3(Y747A) mutation suppressed those effects. These results suggest that perturbation of the beta3 tail membrane-proximal region is associated with talin-mediated integrin activation.

One hundred years ago, we knew very little about biological macromolecules and had no tools available to study their structure. Structural biology is now a mature science. New structures are being solved at an ever-increasing rate and there are important new initiatives to determine all the protein folds that are used by biological systems (structural genomics). This article traces some of the key developments in the field.

SpoIIAA is a key component in the network of interactions that regulate the first sporulation-specific transcription factor, σ F , in Bacillus subtilis . In its unphosphorylated form SpoIIAA is either phosphorylated by or forms a non-covalent complex with SpoIIAB, whereas in its phosphorylated form it is dephosphorylated by SpoIIE. In this work we present NMR studies of the SpoIIAA 2 .SpoIIAB complex and of mutant proteins that are deficient in their ability to interact with SpoIIAB or SpoIIE. The NMR studies of the SpoIIAA 2 .SpoIIAB complex allowed us to define a contiguous patch that is perturbed upon complex formation. By examining the chemical shift perturbations in the mutant proteins we have identified more specific areas that contain residues critical for the SpoIIAB and SpoIIE interactions.

Background: Growth and morphogenesis during development depend both on patterning genes, which assign positional information, and on genes that regulate mechanical forces. The dumpy gene of the fruit fly Drosophila melanogaster is an example of the latter class, with mutant phenotypes affecting size and shape of the limbs, thoracic cuticle, trachea and mouthparts. Results: The genetically complex dumpy locus was found to span over 100 kb and encode a gigantic 2.5 MDa extracellular matrix protein. Dumpy represents an extreme form of modular protein evolution, containing 308 epidermal growth factor (EGF) modules, interspersed with a new module class, DPY, and terminating in a crosslinking zona pellucida domain and membrane anchor sequence. We determined the three-dimensional structure of the DPY module by nuclear magnetic resonance (NMR) spectroscopy and found that it forms a disulphide-stabilised β sheet motif, capable of linking end-to-end with EGF modules to form a fibre. Consistent with its cuticle phenotypes, dumpy is expressed at several sites of cuticle–epidermal cell attachment, including the trachea and the muscle tendon cells, which mediate anchorage of the muscles to the cuticle. Conclusions: The dumpy gene encodes a gigantic extracellular molecule that we predict to be a membrane-anchored fibre of almost a micrometer in length. Insertion and crosslinking of this fibre within the cuticle may provide a strong anchor for the underlying tissue, allowing it to maintain mechanical tension at sites under stress. This would explain its contribution to tissue morphogenesis through its regulation of mechanical properties.

The ninth and tenth type III domains of fibronectin each contain specific cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion. We investigated the relationship between domain-domain orientation and synergistic adhesive activity of the FIII9 and FIII10 pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9 and FIII10 domains. Incremental extensions of the interdomain link between FIII9 and FIII10 reduced the initial cell attachment, but had a much more pronounced effect on the downstream cell adhesion events of spreading and phosphorylation of focal adhesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker. Nuclear magnetic resonance spectroscopy of the wild type and mutant FIII9-10 proteins demonstrated that the structure of the RGD-containing loop is unaffected by domain-domain interactions. We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of these sites. These data have implications for the molecular mechanisms by which integrin-ligand interactions are achieved.

Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation., Author Summary Transmission of signals across the cell membrane is an essential process for all living organisms. Integrins are one example of cell surface receptors (αβ) which, uniquely, form a bidirectional signalling pathway across the membrane. Integrins are crucial for many cellular processes and play key roles in pathological defects such as cardiovascular diseases and cancer. They are activated to a high affinity state by the intracellular protein talin in a process known as ‘inside-out activation’. Despite their importance and the existence of functional and structural data, the mechanism by which talin activates integrin remains elusive. In this study we use a multi-scale computational approach, which combines coarse-grained and atomistic molecular dynamics simulations, to suggest how the formation of the complex between the talin head domain, the cell membrane and the integrin moves the integrin equilibrium towards an active state. Our results show that conformational changes within the talin head domains optimize its interactions with the cell membrane. Upon binding to the integrin, talin facilitates rearrangement of the integrin TM region thus promoting integrin activation. This study also provides a demonstration of the strengths of a computational multi-scale approach in studies of membrane interactions and receptor conformational changes and associated proteins that enable transmembrane signaling.

Background: The fibronectin (FN)-collagen interaction is important for cell adhesion and migration. Results: FN modules 8–9FnI interact with two distinct sites in both chains of collagen I. All six collagen-binding FN modules interact cooperatively with a single collagen site. Conclusion: Collagen I possesses four equipotent sites for FN. Significance: We have mapped FN binding to collagen I and demonstrated the first cooperative interaction., Despite its biological importance, the interaction between fibronectin (FN) and collagen, two abundant and crucial tissue components, has not been well characterized on a structural level. Here, we analyzed the four interactions formed between epitopes of collagen type I and the collagen-binding fragment (gelatin-binding domain (GBD)) of human FN using solution NMR, fluorescence, and small angle x-ray scattering methods. Collagen association with FN modules 8–9FnI occurs through a conserved structural mechanism but exhibits a 400-fold disparity in affinity between collagen sites. This disparity is reduced in the full-length GBD, as 6FnI1–2FnII7FnI binds a specific collagen epitope next to the weakest 8–9FnI-binding site. The cooperative engagement of all GBD modules with collagen results in four broadly equipotent FN-collagen interaction sites. Collagen association stabilizes a distinct monomeric GBD conformation in solution, giving further evidence to the view that FN fragments form well defined functional and structural units.

Interleukin 2 (IL-2) is one of the major cytokines produced by T lymphocytes in response to antigen. It is a potent growth and differentiation factor for several cell-types and is structurally related to the four-helix bundle family of cytokines. Mutation of residue Phe42 to Ala abolishes binding to the alpha chain of the tri-partite IL-2 receptor. The three-dimensional structure of the F42A mutant IL-2 has been calculated by two dimensional NMR methods and compared to a structure of wild-type IL-2 determined by X-ray crystallography. The overall topology of the two structures is the same. The main differences between the structures are within the ill-defined loops connecting the helices and the region of the protein that is believed to interact with the alpha-chain of the receptor. Thus, the mutation of Phe42 to Ala does not perturb the overall three-dimensional structure of IL-2, and does not appear to change the putative binding sites for the beta and gamma chains of the receptor. The structural differences observed in this mutant suggest that the replacement of Phe42 with Ala causes the re-orientation of neighbouring side-chains that are also involved in binding the alpha-chain of the receptor.

Many growth factors and cytokines promote receptor clustering on binding. At least three different protein-protein interaction sites are involved: cytokine-receptor I, cytokine-receptor II and receptor I-receptor II. Although structural data on these complexes are limited, recent structural and mutagenesis studies of the four-helix bundle class of cytokines are clarifying the nature of the complexes formed.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin interactions including those for glycoprotein Ibα and integrins have been mapped to the region comprising FLN domains 16-21. Traditionally, FLN domains have been viewed as independent folding units, arranged in a linear chain joined with flexible linkers. Recent structural findings have shown that consecutive FLNs form more intricate superstructures. The crystal structure of filamin A domains 19-21 (FLNa19-21) revealed that domains 20 and 21 fold together and that the domain interaction can be autoregulatory. The solution structure of domains 18-19 showed a similar domain interaction, whereas domain pair 16-17 has a completely different domain packing mode. In this study, we characterize the domain organization of the FLNa domain sextet 16-21 using NMR spectroscopy. A structure model of this 60-kDa protein has been built using residual dipolar coupling restraints. RDCs and (15)N relaxation data have been used to characterize interdomain motions.

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the (8)F1(9)F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to (8)F1(9)F1 and that UR binding to (8)F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.

Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the beta integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated (15)N-labeled beta3, beta1A, and beta7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of beta3, beta1A, and beta7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of beta integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins.

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains (4-5)FnI and (3)FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes (3)FnIII and disrupts the observed (4-5)FnI-(3)FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655-1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316-326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with beta3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.

THE CD2 antigen is largely restricted to cells of the T-lymphocyte lineage and has been established as an important adhesion molecule in interactions between human T lymphocytes and accessory cells1. In the adhesion reaction, CD2 on T cells binds to LFA-3 on other cells, with binding through domain 1 of CD2 (ref. 2). CD2 can also be a target for the delivery of mitogenic signals to T lymphocytes cultured with combinations of anti-CD2 antibodies3,4. Two predictions that are contradictory have been made for the structure of CD2 domain 1. One suggests an immunoglobulin (Ig) fold, on the basis of sequence patterns conserved in the Ig-superfamily (IgSF)5, whilst the other proposes a pattern of alternating α-helices and β-strands, on the basis of secondary structure predictions6. Thus CD2 domain 1 is an important test case for the validity of IgSF assignments based on sequence patterns. We report here the expression of domain 1 of rat CD2 in an Escherichia coli expression system and have determined a low-resolution solution structure by NMR spectroscopy.

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro5 and Pro19 and binds to the CD face of the IgFLNa21 β-sandwich. This interaction is similar to the previously characterized β7 integrin-IgFLNa21 interaction and migfilin and integrin β tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.

Integrins are heterodimeric membrane-spanning adhesion receptors that are essential for a wide range of biological functions. Control of integrin conformational states is required for bidirectional signalling across the membrane. Key components of this control mechanism are the transmembrane and cytoplasmic domains of the alpha and beta subunits. These domains are believed to interact, holding the integrin in the inactive state, while inside-out integrin activation is accompanied by domain separation. Although there are strong indications for domain interactions, the majority of evidence is insufficient to precisely define the interaction interface. The current best model of the complex, derived from computational calculations with experimental restraints, suggests that integrin activation by the cytoplasmic protein talin is accomplished by steric disruption of the alpha/beta interface. Better atomic-level resolution structures of the alpha/beta transmembrane/cytoplasmic domain complex are still required for the resting state integrin to corroborate this. Integrin activation is also controlled by competitive interactions involving the cytoplasmic domains, particularly the beta-tails. The concept of the beta integrin tail as a focal adhesion interaction 'hub' for interactions and regulation is discussed. Current efforts to define the structure and affinity of the various complexes formed by integrin tails, and how these interactions are controlled, e.g. by phosphorylation and localization, are described.

The bioenergetics of two extreme halophiles, Halobacterium halobium and Halobacterium marismortui, have been investigated by respiratory studies and in vivo nuclear magnetic resonance (NMR) spectroscopy. The phosphorus resonances of both species were very broad under anaerobic conditions, but sharpened somewhat on initiation of respiration and considerably upon cooling; changes attributable in part to slow chemical exchange processes. Transmembrane pH gradients (ΔpH) of H. marismortui were somewhat lower than those of H. halobium. Uncoupling agents caused ATP to disappear and inhibited respiration in both species, but dissipated ΔpH at best only partially. Uptake of Mn2+ by H. halobium was unaffected by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), whereas H. marismortui appeared not to transport the ion, even in the absence of uncoupler. FCCP caused a slow efflux of K+ from H. halobium, but no K+ was lost from H. marismortui. These findings are discussed with particular reference to the intracellular environments of the two organisms.

A new protocol for measuring cellular uptake of dipeptides was developed in which the problem of peptide hydrolysis is obviated by introduction into the cell suspension of a membrane-permeant peptidase inhibitor. The uptake of unlabelled dipeptide is readily monitored so long as some analytical technique is available for measuring the intracellular peptide concentration; in this study we used n.m.r. spectroscopy. Using this protocol, we demonstrated that dipeptide uptake by human erythrocytes occurs by simple diffusion through the lipid bilayer and not via a high-capacity protein-mediated transport system. Substantiating evidence includes demonstration that: (a) the fluxes are slow compared with known protein-mediated transport processes in human erythrocytes; (b) the uptake is not stereospecific; (c) the uptake does not display saturation kinetics; (d) the fluxes are significantly enhanced by butanol; (e) a distinct correlation exists between the size-corrected permeability coefficients of the dipeptides and their calculated n-octanol/water partition coefficients. It is calculated that under normal physiological conditions the diffusive fluxes of circulating plasma peptides into human erythrocytes are too small for these cells to play a significant role in dipeptide catabolism.

Summary The solution structures of the homologous growth factors hEGF and hTGF-α, have been determined independently from high resolution nuclear magnetic resonance (NMR) data. A model of the insulin-like growth factor structure based on insulin coordinates (Blundell et al. (1978) Proc natn. Acad. Sci. U.S.A.75, 180–184), has also been refined using molecular dynamics simulations with NMR-determined restraints. Knowledge of these structures, together with known sequences of other homologous proteins and experiments with site-specific residue changes, allows predictions to be made about growth factor residues which might be involved in the receptor–ligand interfaces.

(1) Chloroplast F1-ATPase (CF1) was labelled on both a single tyrosine and at least two cysteine residues by 4-chloro-7-nitrobenzofurazan (Nbf-Cl). The ATPase activity was inhibited by 80–90%. Prior treatment of latent CF1 with N-ethylmaleimide (NEM), to block accessible thiol groups, reduced the Nbf-cysteine modification to the equivalent of one residue per enzyme molecule. Only one tyrosine residue, identified here as tyrosine 328 on one beta subunit, is modified by Nbf-Cl. This is analogous to the tyrosine residue which reacts with Nbf-Cl on the related enzyme from mitochondria (MF1). (2) 31P and 1H nuclear magnetic resonance (NMR) studies of CF1 identified a single tight binding site for the Ca2+ complex of the ATP analogue 5′-adenylylimidodiphosphate (AMPPNP) on each of latent, trypsin activated, and NEM modified CF1. No NMR signals could be detected from from this site. Tight binding of Ca-AMPPNP to latent and trypsin activated CF1 was also inferred from its low inhibition constant (Ki ≈ 12 μM). NMR also showed that addition of excess Ca-AMPPNP to both latent and NEM-labelled CF1 displaced one tightly bound adenine nucleotide. The 31P signals from nucleotides ADP or ATP bound at this single tight site were too broad to be observed but could readily be detected after denaturation of the enzyme with detergent. (3) In contrast to the active forms of CF1, the Nbf-Cl modified enzyme was found to have one ADP and one ATP bound. 31P-NMR signals from the ADP, but not the ATP, could be detected. These signals had the linewidths expected for a bound ligand. In the presence of excess Ca-AMPPNP the bound nucleotide slowly dissociated from the Nbf labelled enzyme at a rate which correlated with loss of the Nbf group from tyrosine 328. This was accompanied by a loss of signal intensity equivalent to one mol of Ca-AMPPNP per mol enzyme.

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.

The extracellular matrix (ECM) is a highly organized multimolecular structure, essential for life in higher organisms. Although substantial high-resolution structural information is available for relatively small fragments of ECM components, the inherent difficulty in preparing and analyzing samples of large, fibrous polymers impedes structural efforts. Here, we review recent advances in understanding the structure of three important ECM components: collagen, fibrillin and fibronectin. Emphasis is placed on the key role of intermolecular interactions in assembling larger, microm scale, structures.

Cell migration is crucial for many functions in metozoan organisms, such as embryonic development, wound repair, cancer invasion and immunity. Migration involves actin-nucleation based membrane protrusion, new focal adhesion formation and focal adhesion disassembly. Calpain is a Ca2+-dependent enzyme that localizes to focal adhesion, cleaves a large number of adhesion proteins and is believed to be involved in disassembly of focal adhesions. An elegant experiment by Franco et al. 2004 showed that calpain cleavage of talin is a rate limiting process in adhesion disassembly. One of the two calpain isoforms, μ-calpain, is activated by micromolar Ca2+. It has been demonstrated that calcium fluxes are capable of activating μ-calpain, which is required for cell motility of keratinocytes. Stretch-activated Ca2+ channels are major regulators of cell migration and are believed to be involved in Ca2+-dependent μ-calpain activation. However, the mechanism of the Ca2+-dependent μ-calpain activation remains poorly characterized. Here we develop a model for Ca2+ dynamics and Ca2+-dependent μ-calpain activation. The model includes a number of Ca2+dependent proteins: Ca2+-dependent PLCδ (and also Ca2+ independent PLC), IP3 channels on the ER membrane, the calcium pumps, and stretch activated Ca2+ channels. We show that stretch activated channels may work as a switch, turning Ca2+ oscillations on or off. Increases in calcium activate μ-calpain, causing μ-calpain activity impulses. The amplitude, frequency and duration of the impulses have been studied as a function of the system's components, in particular as a function of Ca2+dependent and independent PLC activities. The implications for Ca2+-dependent μ-calpain activity in adhesion turnover are discussed. from BioSysBio: Bioinformatics and Systems Biology Conference Edinburgh, UK, 14–15 July 2005

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.

Integrins mediate cell adhesion and are essential receptors for the development and functioning of multicellular organisms. Integrin activation is known to require both ligand and talin binding and to correlate with cluster formation but the activation mechanism and precise roles of these processes are not yet resolved. Here mathematical modeling, with known experimental parameters, is used to show that the binding of a stabilizing factor, such as talin, is alone insufficient to enable ligand-dependent integrin activation for all observed conditions; an additional positive feedback is required., in press in Bulletin of Mathematical Biology

The gelatin (denatured collagen) binding domain of the extracellular matrix protein fibronectin contains three potential N-glycosylation sites. Complete deglycosylation of this domain is known to reduce the thermal stability of the eighth type 1 (8F1) module. We have conducted a site-specific analysis of the structural and functional consequences of N-linked glycosylation in the 8F19F1 module pair. Three glycoforms have been identified by mass spectrometry and nuclear magnetic resonance spectroscopy. Chemical shift differences between the glycoforms have revealed an intimate interaction between one N-linked sugar and the polypeptide that is critical for gelatin binding, as shown by affinity chromatography.