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We demonstrate an automated microfluidic nuclear magnetic resonance (NMR) system that quantitatively characterizes protein-ligand interactions without user intervention and with minimal sample needs through protein-detected heteronuclear 2D NMR spectroscopy. Quantitation of protein-ligand interactions is of fundamental importance to the understanding of signaling and other life processes. As is well-known, NMR provides rich information both on the thermodynamics of binding and on the binding site. However, the required titrations are laborious and tend to require large amounts of sample, which are not always available. The present work shows how the analytical power of NMR detection can be brought in line with the trend of miniaturization and automation in life science workflows.

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein–protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.

We present a microfluidic platform that allows in operando nuclear magnetic resonance (NMR) observation of serial mixing experiments. Gradually adding one reagent to another is a fundamental experimental modality, widely used to quantify equilibrium constants, for titrations, and in chemical kinetics studies. NMR provides a non-invasive means to quantify concentrations and to follow structural changes at the molecular level as a function of exchanged volume. Using active pneumatic valving on the microfluidic device directly inside an NMR spectrometer equipped with a transmission-line NMR microprobe, the system allows injection of aliquots and in situ mixing in a sample volume of less than 10 μL.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 process N-terminally extended antigenic precursors for optimal loading onto major histocompatibility complex class I (MHC I) molecules. We and others have demonstrated that ERAP1 processes peptides bound to MHC I, but the underlying mechanism is unknown. To this end, we utilized single-chain trimers (SCT) of the ovalbumin-derived epitope SIINFEKL (SL8) tethered to the H2-Kb MHC I determinant from mouse and introduced three substitutions, E63A, K66A, and W167A, at the A-pocket of the peptide-binding groove in the MHC I heavy chain, which interact with the N termini of peptides. These variants significantly decreased SL8-presenting SCT at the cell surface in the presence of ERAP1, but did not affect overall SCT expression, indicating that ERAP1 trims the SL8 N terminus. Comparison of the X-ray crystal structures of WT and three variant SCTs revealed only minor perturbations of the peptide-binding domain in the variants. However, molecular dynamics simulations suggested that SL8 can dissociate partially within a sub-microsecond timescale, exposing its N terminus to the solvent. We also found that the C terminus of MHC I–bound SL8 remains deeply buried in the F-pocket of MHC I. Furthermore, free-energy calculations revealed that the three SCT variants exhibit lower free-energy barriers of N terminus dissociation than the WT Kb. Taken together, our results are consistent with a previously observed model in which the partial dissociation of bound peptides from MHC I exposes their N terminus to trimming by ERAP1, whereas their C terminus is anchored at the F-pocket.

Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca(2+)/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis.

Major histocompatibility complex class I molecules (MHC I) help protect jawed vertebrates by binding and presenting immunogenic peptides to cytotoxic T lymphocytes. Peptides are selected from a large diversity present in the endoplasmic reticulum. However, only a limited number of peptides complement the polymorphic MHC specificity determining pockets in a way that leads to high-affinity peptide binding and efficient antigen presentation. MHC I molecules possess an intrinsic ability to discriminate between peptides, which varies in efficiency between allotypes, but the mechanism of selection is unknown. Elucidation of the selection mechanism is likely to benefit future immune-modulatory therapies. Evidence suggests peptide selection involves transient adoption of alternative, presumably higher energy conformations than native peptide–MHC complexes. However, the instability of peptide-receptive MHC molecules has hindered characterization of such conformational plasticity. To investigate the dynamic nature of MHC, we refolded MHC proteins with peptides that can be hydrolyzed by UV light and thus released. We compared the resultant peptide-receptive MHC molecules with non-hydrolyzed peptide-loaded MHC complexes by monitoring the exchange of hydrogen for deuterium in solution. We found differences in hydrogen–deuterium exchange between peptide-loaded and peptide-receptive molecules that were negated by the addition of peptide to peptide-receptive MHC molecules. Peptide hydrolysis caused significant increases in hydrogen–deuterium exchange in sub-regions of the peptide-binding domain and smaller increases elsewhere, including in the α3 domain and the non-covalently associated β2-microglobulin molecule, demonstrating long-range dynamic communication. Comparing two MHC allotypes revealed allotype-specific differences in hydrogen–deuterium exchange, consistent with the notion that MHC I plasticity underpins peptide selection.

The calcium-binding epidermal growth factor-like (cbEGF) module and the transforming growth factor β-binding protein-like (TB) module are the two major structural motifs found in fibrillin-1, the extracellular matrix (ECM) protein defective in the Marfan syndrome (MFS). An MFS-causing mutation, N2144S, which removes a calcium ligand in cbEGF32, does not detectably affect fibrillin-1 biosynthesis, rate of secretion, processing, or deposition of reducible fibrillin-1 into the ECM. Since the residue at position 2144 is normally engaged in calcium ligation, it is unable to mediate intermolecular interactions. We have shown previously that this mutation does not affect the folding properties of the TB or cbEGF domains in vitro, but does decrease calcium-binding in cbEGF and TB-cbEGF domain constructs. Here, we use NMR spectroscopy to probe the effects of the N2144S mutation on backbone dynamic properties of TB6-cbEGF32. Analysis of the backbone 15N relaxation data of wild-type TB6-cbEGF32 has revealed a flexible inter-domain linkage. Parallel dynamics analysis of the N2144S mutant has shown increased flexibility in the region joining the two domains as well as in the calcium-binding site at the N terminus of cbEGF32. This research demonstrates that a small change in peptide backbone flexibility, which does not enhance proteolytic susceptibility of the domain pair, is associated with an MFS phenotype. Flexibility of the TB-cbEGF linkage is likely to contribute to the biomechanical properties of fibrillin-rich connective tissue microfibrils, and may play a role in the microfibril assembly process.

The lone (1)F2(2)F2 modular pair of fibronectin is found in the collagen-binding region. This exclusive localization suggests the (1)F2(2)F2 pair plays an important role in the recognition of collagen. However, no information is currently available about the interaction between the two F2 modules and, thus, the orientation of their putative collagen-binding sites with respect to one another. Comparison of a variety of high-resolution NMR parameters from the F2 modules in isolation and the (1)F2(2)F2 pair was used to establish the extent of interaction between the F2 modules in the pair. Chemical shifts of the F2 modules and the (1)F2(2)F2 pair indicate that the structures of the modules are preserved in the pair and that, with the exception of the covalent linkage, they do not interact. (15)N NMR relaxation data identify significant motion occurring in the linker region of the (1)F2(2)F2 pair, and analyses of the anisotropic diffusion properties of the (1)F2(2)F2 pair are consistent with the modules in the F2 pair tumbling independent of one another.

Protein synthesis, and especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalysed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent ?-kinase.Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously-known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalysed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally-conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the CaM-stimulated activity of eEF2K. Neuronal cells depend on oxygen and eEF2K helps to protect them from hypoxia.eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly-vascularised solid tumours.

The protein calexcitin was originally identified in molluscan photoreceptorneurons as a 20 kDa molecule which was up-regulated and phosphorylatedfollowing a Pavlovian conditioning protocol. Subsequent studies showed thatcalexcitin regulates the voltage-dependent potassium channel and the calciumdependentpotassium channel as well as causing the release of calcium ions fromthe endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystalstructure of calexcitin from the squid Loligo pealei showed that the fold issimilar to that of another signalling protein, calmodulin, the N- and C-terminaldomains of which are known to separate upon calcium binding, allowinginteractions with the target protein. Phosphorylation of calexcitin causes it totranslocate to the cell membrane, where its effects on membrane excitability areexerted and, accordingly, L. pealei calexcitin contains two protein kinase Cphosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimicphosphorylation of the protein were introduced and crystal structures of thecorresponding single and double mutants were determined, which suggest thatthe C-terminal phosphorylation site (Thr188) exerts the greatest effects onthe protein structure. Extensive NMR studies were also conducted, whichdemonstrate that the wild-type protein predominantly adopts a more openconformation in solution than the crystallographic studies have indicated and,accordingly, normal-mode dynamic simulations suggest that it has considerablygreater capacity for flexible motion than the X-ray studies had suggested. Likecalmodulin, calexcitin consists of four EF-hand motifs, although only the firstthree EF-hands of calexcitin are involved in binding calcium ions; the C-terminalEF-hand lacks the appropriate amino acids. Hence, calexcitin possesses twofunctional EF-hands in close proximity in its N-terminal domain and onefunctional calcium site in its C-terminal domain. There is evidence that theprotein has two markedly different affinities for calcium ions, the weaker ofwhich is most likely to be associated with binding of calcium ions to the proteinduring neuronal excitation. In the current study, site-directed mutagenesis hasbeen used to abolish each of the three calcium-binding sites of calexcitin, andthese experiments suggest that it is the single calcium-binding site in theC-terminal domain of the protein which is likely to have a sensory role in theneuron.

The catalytic activity of Src-family kinases is regulated by association with its SH3 and SH2 domains. Activation requires displacement of intermolecular contacts by SH3/SH2 binding ligands resulting in dissociation of the SH3 and SH2 domains from the kinase domain. To understand the contribution of the SH3-SH2 domain pair to this regulatory process, the binding of peptides derived from physiologically relevant SH2 and SH3 interaction partners was studied for Lck and its relative Fyn by NMR spectroscopy. In contrast to Fyn, activating ligands do not induce communication between SH2 and SH3 domains in Lck. This can be attributed to the particular properties of the Lck SH3-SH2 linker which is shown to be extremely flexible thus effectively decoupling the behavior of the SH3 and SH2 domains. Measurements on the SH32 tandem from Lck further revealed a relative domain orientation that is distinctly different from that found in the Lck SH32 crystal structure and in other Src kinases. These data suggest that flexibility between SH2 and SH3 domains contributes to the adaptation of Src-family kinases to specific environments and distinct functions.

Formation of infectious HIV-1 involves assembly of Gag polyproteins into immature particles and subsequent assembly of mature capsids after proteolytic disassembly of the Gag shell. We report a 12-mer peptide, capsid assembly inhibitor (CAI), that binds the capsid (CA) domain of Gag and inhibits assembly of immature- and mature-like capsid particles in vitro. CAI was identified by phage display screening among a group of peptides with similar sequences that bind to a single reactive site in CA. Its binding site was mapped to CA residues 169-191, with an additional contribution from the last helix of CA. This result was confirmed by a separate X-ray structure analysis showing that CAI inserts into a conserved hydrophobic groove and alters the CA dimer interface. The CAI binding site is a new target for antiviral development, and CAI is the first known inhibitor directed against assembly of immature HIV-1.

Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [(1)H]-(15)N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.

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.

Highlights • MHC class I alleles vary in their intrinsic ability to select optimal peptides. • Ability of MHC to self-assemble is inversely correlated with dependence on tapasin. • Variation in peptide selector function correlates with the plasticity of empty MHC. • Increased plasticity of empty MHC I allows more efficient peptide selector function. • Co-ordinated domain–domain movements contribute to determine plasticity., Major histocompatibility complex class I (MHC I) proteins provide protection from intracellular pathogens and cancer via each of a cell's MHC I molecules binding and presenting a peptide to cytotoxic T lymphocytes. MHC I genes are highly polymorphic and can have significant diversity, with polymorphisms predominantly localised in the peptide-binding groove where they can change peptide-binding specificity. However, polymorphic residues may also determine other functional properties, such as how dependent MHC I alleles are on the peptide-loading complex for optimal acquisition of peptide cargo. We describe how differences in the peptide-binding properties of two MHC I alleles correlates with altered conformational flexibility in the peptide-empty state. We hypothesise that plasticity is an intrinsic property encoded by the protein sequence, and that co-ordinated movements of the membrane-proximal and membrane-distal domains collectively determines how dependent MHC I are on the peptide-loading complex for efficient assembly with high affinity peptides.

The regulatory domains of Src family kinases SH3 and SH2 suppress Src activity when bound to the catalytic domain. Here, the isolated SH3-SH2 fragment from the Src family member Fyn (FynSH32) is studied by NMR. The properties of this fragment are expected to be similar to the domains in the active state, where they are dissociated from the catalytic domain. Crosscommunication between SH3 and SH2 of FynSH32, measured by chemical shift perturbation, was found to be small. Diffusion and alignment anisotropy measurements showed that SH3 and SH2 of peptide-bound FynSH32 are significantly coupled but still exhibit some interdomain flexibility. The observed average domain orientation indicates that a large SH3-SH2 domain closure is required to reach the inactive state. The implications of these results for Src regulation are discussed.

Over expression of proteins in E. coli frequently results in the production of inclusion bodies. Although β(2) -microglobulin frequently forms fibrillar structures, our studies reveal significant differences between the protein in fibrils and inclusion bodies. This suggests that the formation of fibrils in inclusion bodies is dependent on the propensity of the protein to form fibrillar structures.

Highlights • The expression of matrilin VWA domains was improved. • The proper folding was monitored by NMR and CD spectroscopy. • Binding properties were analyzed by surface plasmon resonance. • Differences between bacterially and eukaryotically expressed domains were detected. • We recommend eukaryotic expression for structural analysis., VWA domains are the predominant independent folding units within matrilins and mediate protein–protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems.

Background: From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. Results: Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. Conclusions: The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.

Background: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA–protein interactions remains poorly understood. Results: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA 4 –HA 16 ). All oligomers bound (except HA 4 ) with K d values ranging from 0.2–0.5 μM at 25°C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan–protein interactions. The HA 8 recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the β4/β5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. Conclusions: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.

Calcium binding (cb) epidermal growth factor-like (EGF) domains are found in a wide variety of extracellular proteins with diverse functions. In several proteins, including the fibrillins (1 and 2), the low-density lipoprotein receptor, the Notch receptor and related molecules, these domains are organised as multiple tandem repeats. The functional importance of calcium-binding by EGF domains has been underscored by the identification of missense mutations associated with defective calcium-binding, which have been linked to human diseases. Here, we present 15N backbone relaxation data for a pair of cbEGF domains from fibrillin-1, the defective protein in the Marfan syndrome. The data were best fit using a symmetric top model, confirming the extended conformation of the cbEGF domain pair. Our data demonstrate that calcium plays a key role in stabilising the rigidity of the domain pair on the pico- to millisecond time-scale. Strikingly, the most dynamically stable region of the construct is centred about the domain interface. These results provide important insight into the properties of intact fibrillin-1, the consequences of Marfan syndrome causing mutations, and the ultrastructure of fibrillins and other extracellular matrix proteins.

Major histocompatibility complex class I molecules (MHC I) present peptides to cytotoxic T-cells at the surface of almost all nucleated cells. The function of MHC I molecules is to select high affinity peptides from a large intracellular pool and they are assisted in this process by co-factor molecules, notably tapasin. In contrast to mammals, MHC homozygous chickens express a single MHC I gene locus, termed BF2, which is hypothesised to have co-evolved with the highly polymorphic tapasin within stable haplotypes. The BF2 molecules of the B15 and B19 haplotypes have recently been shown to differ in their interactions with tapasin and in their peptide selection properties. This study investigated whether these observations might be explained by differences in the protein plasticity that is encoded into the MHC I structure by primary sequence polymorphisms. Furthermore, we aimed to demonstrate the utility of a complimentary modelling approach to the understanding of complex experimental data. Combining mechanistic molecular dynamics simulations and the primary sequence based technique of statistical coupling analysis, we show how two of the eight polymorphisms between BF2*15∶01 and BF2*19∶01 facilitate differences in plasticity. We show that BF2*15∶01 is intrinsically more plastic than BF2*19∶01, exploring more conformations in the absence of peptide. We identify a protein sector of contiguous residues connecting the membrane bound α3 domain and the heavy chain peptide binding site. This sector contains two of the eight polymorphic residues. One is residue 22 in the peptide binding domain and the other 220 is in the α3 domain, a putative tapasin binding site. These observations are in correspondence with the experimentally observed functional differences of these molecules and suggest a mechanism for how modulation of MHC I plasticity by tapasin catalyses peptide selection allosterically.

The nuclear magnetic resonance structure of a covalently linked pair of calcium-binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, the protein defective in the Marfan syndrome, is described. The two domains are in a rigid, rod-like arrangement, stabilized by interdomain calcium binding and hydrophobic interactions. We propose a model for the arrangement of fibrillin monomers in microfibrils that reconciles structural and antibody binding data, and we describe a set of disease-causing mutations that provide the first clues to the specificity of cbEGF interactions. The residues involved in stabilizing the domain linkage are highly conserved in fibrillin, fibulin, thrombomodulin, and the low density lipoprotein receptor. We propose that the relative orientation of tandem cbEGF domains in these proteins is similar, but that in others, including Notch, pairs adopt a completely different conformation.

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

The three dimensional structure of the neuronal calcium-sensor protein calexcitin from Loligo pealei has been determined by X-ray analysis at a resolution of 1.8 A. Calexcitin is up-regulated following Pavlovian conditioning and has been shown to regulate potassium channels and the ryanodine receptor. Thus calexcitin is implicated in neuronal excitation and plasticity. The overall structure is predominantly helical and compact with a pronounced hydrophobic core between the N- and C-terminal domains of the molecule. The structure consists of four EF-hand motifs although only the first three EF hands are involved in binding calcium ions; the C-terminal EF-hand lacks the amino acids required for calcium binding. The overall structure is quite similar to that of the sarcoplasmic calcium binding protein from Amphioxus although the sequence identity is very low at 31 %. The structure shows that the two amino acids of calexcitin phosphorylated by protein kinase C are close to the domain interface in three dimensions and thus phosphorylation is likely to regulate the opening of the domains that is probably required for binding to target proteins. There is evidence that calexcitin is a GTPase and the residues which have been implicated by mutagenesis in its GTPase activity are in a short but highly conserved region of 3-10 helix close to the C-terminus. This helix resides in a large loop that is partly sandwiched between the N- and C-terminal domains suggesting that GTP binding may also require or may cause domain opening. The structure possesses a pronounced electropositive crevice, in the vicinity of the 3-10 helix, that might provide an initial docking site for the triphosphate group of GTP. These findings elucidate a number of the reported functions of calexcitin with implications for neuronal signalling.

Integrins are an important family of signaling receptors that mediate diverse cellular processes. The binding of the abundant extracellular matrix ligand fibronectin to integrins alpha(5)beta(1) and alpha(v)beta(3) is known to depend upon the Arg-Gly-Asp (RGD) motif on the tenth fibronectin FIII domain. The adjacent ninth FIII domain provides a synergistic effect on RGD-mediated integrin alpha(5)beta(1) binding and downstream function. The precise molecular basis of this synergy remains elusive. Here we have dissected further the function of FIII9 in integrin binding by analyzing the biological activity of the FIII9-10 interdomain interface variants and by determining their structural and dynamic properties in solution. We demonstrate that the contribution of FIII9 to both alpha(5)beta(1) and alpha(v)beta(3) binding and downstream function critically depends upon the interdomain tilt between the FIII9 and FIII10 domains. Our data suggest that modulation of integrin binding by FIII9 may arise in part from its steric properties that determine accessibility of the RGD motif. These findings have wider implications for mechanisms of integrin-ligand binding in the physiological context.

We present NMR structural and dynamics analysis of the putative ligand binding region of human Notch-1, comprising EGF-like domains 11–13. Functional integrity of an unglycosylated, recombinant fragment was confirmed by calcium-dependent binding of tetrameric complexes to ligand-expressing cells. EGF modules 11 and 12 adopt a well-defined, rod-like orientation rigidified by calcium. The interdomain tilt is similar to that found in previously studied calcium binding EGF pairs, but the angle of twist is significantly different. This leads to an extended double-stranded β sheet structure, spanning the two EGF modules. Based on the conservation of residues involved in interdomain hydrophobic packing, we propose this arrangement to be prototypical of a distinct class of EGF linkages. On this premise, we have constructed a model of the 36 EGF modules of the Notch extracellular domain that enables predictions to be made about the general role of calcium binding to this region.

The combination of chemical shift, residual dipolar coupling, and backbone relaxation data can be used to characterize the nature of a domain interface in a multidomain protein. Comparison of the parameters obtained from isolated domains and domain pairs provides insight into the composition of the interface as well as into interdomain dynamics. The interface between the 13th and 14th F3 module from fibronectin is used as an example.

The combination of chemical shift, residual dipolar coupling, and backbone relaxation data can be used to characterize the nature of a domain interface in a multidomain protein. Comparison of the parameters obtained from isolated domains and domain pairs provides insight into the composition of the interface as well as into interdomain dynamics. The interface between the 13th and 14th F3 module from fibronectin is used as an example.

Fibrillin-1 is a mosaic protein mainly composed of 43 calcium binding epidermal growth factor-like (cbEGF) domains arranged as multiple, tandem repeats. Mutations within the fibrillin-1 gene cause Marfan syndrome (MFS), a heritable disease of connective tissue. More than 60% of MFS-causing mutations identified are localized to cbEGFs, emphasizing that the native properties of these domains are critical for fibrillin-1 function. The cbEGF12-13 domain pair is within the longest run of cbEGFs, and many mutations that cluster in this region are associated with severe, neonatal MFS. The NMR solution structure of Ca(2+)-loaded cbEGF12-13 exhibits a near-linear, rod-like arrangement of domains. This observation supports the hypothesis that all fibrillin-1 (cb)EGF-cbEGF pairs, characterized by a single interdomain linker residue, possess this rod-like structure. The domain arrangement of cbEGF12-13 is stabilized by additional interdomain packing interactions to those observed for cbEGF32-33, which may help to explain the previously reported higher calcium binding affinity of cbEGF13. Based on this structure, a model of cbEGF11-15 that encompasses all known neonatal MFS missense mutations has highlighted a potential binding region. Backbone dynamics data confirm the extended structure of cbEGF12-13 and lend support to the hypothesis that a correlation exists between backbone flexibility and cbEGF domain calcium affinity. These results provide important insight into the potential consequences of MFS-associated mutations for the assembly and biomechanical properties of connective tissue microfibrils.

The regulatory domains of Src family kinases SH3 and SH2 suppress Src activity when bound to the catalytic domain. Here, the isolated SH3-SH2 fragment from the Src family member Fyn (FynSH32) is studied by NMR. The properties of this fragment are expected to be similar to the domains in the active state, where they are dissociated from the catalytic domain. Crosscommunication between SH3 and SH2 of FynSH32, measured by chemical shift perturbation, was found to be small. Diffusion and alignment anisotropy measurements showed that SH3 and SH2 of peptide-bound FynSH32 are significantly coupled but still exhibit some interdomain flexibility. The observed average domain orientation indicates that a large SH3-SH2 domain closure is required to reach the inactive state. The implications of these results for Src regulation are discussed.

The regulatory fragment of Src kinases, comprising Src homology (SH) 3 and SH2 domains, is responsible for controlled repression of kinase activity. We have used a multidisciplinary approach involving crystallography, NMR, and isothermal titration calorimetry to study the regulatory fragment of Fyn (FynSH32) and its interaction with a physiological activator: a fragment of focal adhesion kinase that contains both phosphotyrosine and polyproline motifs. Although flexible, the preferred disposition of SH3 and SH2 domains in FynSH32 resembles the inactive forms of Hck and Src, differing significantly from LckSH32. This difference, which results from variation in the SH3-SH2 linker sequences, will affect the potential of the regulatory fragments to repress kinase activity. This surprising result implies that the mechanism of repression of Src family members may vary, explaining functional distinctions between Fyn and Lck. The interaction between FynSH32 and focal adhesion kinase is restricted to the canonical SH3 and SH2 binding sites and does not affect the dynamic independence of the two domains. Consequently, the interaction shows no enhancement by an avidity effect. Such an interaction may have evolved to gain specificity through an extended recognition site while maintaining rapid dissociation after signaling.

The transforming growth factor beta (TGF-beta) binding protein-like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin-1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin-1 has revealed the existence of two stable conformers that differ in the isomerization states of two proline residues. Unusually, the two isoforms do not readily interconvert and are stable on the time scale of milliseconds. We have computed independent structures of the major and minor conformers of TB6 to assess how the domain fold adjusts to incorporate alternatively cis- or trans-prolines. Based on previous observations, it has been suggested that multiple conformers can only be accommodated in flexible regions of protein structure. In contrast, P22, which exists in trans in the major form and cis in the minor form of TB6, is in a rigid region of the domain, which is confirmed by backbone dynamics measurements. Overall, the structures of the major and minor conformers are similar. However, the secondary structure topologies of the two forms differ as a direct consequence of the changes in proline conformation.

The secondary structure and backbone dynamics of the cytokine, human granulocyte colony-stimulating factor (hG-CSF) have been determined by heteronuclear nuclear magnetic resonance (NMR) techniques. Virtually complete NH, C alpha H, C beta H 15N, 13C alpha, and 13C beta assignment of the 175-residue recombinant protein, methionyl-[Cys-17-Ser]-hG-CSF, was achieved by use of three-dimensional (3D) heteronuclear 1H-15N and triple-resonance 1H-15N-13C experiments. Spectra recorded at 750 MHz aided the assignment of severely overlapped regions. The structures of G-CSF from several species have recently been determined by X-ray diffraction [Hill, C. P., Osslund, T. D., & Eisenberg, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171; Lovejoy, B., Cascio, D., & Eisenberg, D. (1993) J. Mol. Biol. 234, 640-653]. Like several cytokines, hG-CSF has a four-helix topology (A-D) with overhand loop connections, but with an additional helical segment (A') identified in the connection between helix A and helix B. The solution-state determination of the secondary structure is based on short- and medium-range NOEs, backbone J-couplings, and NH exchange data and is corroborated by 13C alpha secondary shifts. The helices are defined as follows: A, 10-38; A',44-53; B, 71-91; C, 102-123; D, 143-172. The dynamics of the amide backbone resonances, investigated using 1H-15N heteronuclear NMR, indicate a rigid protein core with some increased mobility in the AB loop and more pronounced mobility in the CD loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary Von Willebrand factor A (VWA) domains are versatile protein interaction domains with N and C termini in close proximity placing spatial constraints on overall protein structure. The 1.2 Å crystal structures of a collagen VI VWA domain and a disease-causing point mutant show C-terminal extensions that place the N and C termini at opposite ends. This allows a “beads-on-a-string” arrangement of multiple VWA domains as observed for ten N-terminal domains of the collagen VI α3 chain. The extension is linked to the core domain by a salt bridge and two hydrophobic patches. Comparison of the wild-type and a muscular dystrophy-associated mutant structure identifies a potential perturbation of a protein interaction interface and indeed, the secretion of mutant collagen VI tetramers is affected. Homology modeling is used to locate a number of disease-associated mutations and analyze their structural impact, which will allow mechanistic analysis of collagen-VI-associated muscular dystrophy phenotypes., Highlights • The structure of a VWA domain (N5) of collagen VI at 1.2 Å is presented • N and C termini of the domain are at opposite ends • The structure with a myopathy-causing mutation shows altered interaction interface • The impact of mutations in collagen VI VWA domains was analyzed, Becker et al. describe a crystal structure of a von Willebrand factor A (VWA) domain, which has a C-terminal extension that places the N- and C-termini at opposite ends not seen before for A domains. This allows a “beads-on-a-string” arrangement of multiple VWA domains as observed for the collagen VI α3 chain.

International audience; Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.