Pathways by which interleukin 17 induces articular cartilage breakdown in vitro and in vivo.

Overexpression of interleukin (IL-)17 has recently been shown to be associated with a number of pathological conditions. Because IL-17 is found at high levels in the synovial fluid surrounding cartilage in patients with inflammatory arthritis, the present study determined the direct effect of IL-17 on articular cartilage. As shown herein, IL-17 was a direct and potent inducer of matrix breakdown and an inhibitor of matrix synthesis in articular cartilage explants. These effects were mediated in part by leukemia inhibitory factor (LIF), but did not depend on interleukin-1 activity. The mechanism whereby IL-17 induced matrix breakdown in cartilage tissue appeared to be due to stimulation of activity of aggrecanase(s), not matrix metalloproteinase(s). However, IL-17 upregulated expression of matrix metalloproteinase(s) in chondrocytes cultured in monolayer. In vivo, IL-17 induced a phenotype similar to inflammatory arthritis when injected into the intra-articular space of mouse knee joints. Furthermore, a related protein, IL-17E, was found to have catabolic activity on human articular cartilage. This study characterizes the mechanism whereby IL-17 acts directly on cartilage matrix turnover. Such findings have important implications for the treatment of degenerative joint diseases such as arthritis.

IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding.

The proinflammatory cytokine interleukin 17 (IL-17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL-17 homolog, IL-17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL-6, IL-8 and granulocyte colony-stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL-17F reveals that IL-17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical "knot" structure. IL-17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL-17s and neurotrophins with respect to receptor recognition.

A novel family of hairpin peptides that inhibit IgE activity by binding to the high-affinity IgE receptor.

A family of structured peptides that bind to FcepsilonRIalpha, the alpha-chain of the high-affinity receptor for IgE, has been identified. Binding selections using FcepsilonRIalpha and polyvalent peptide-phage libraries yielded a dominant 18-residue peptide-phage clone, as well as related sequences that did not resemble fragments of IgE. Synthetic peptides based on these sequences inhibited IgE binding to its receptor, and were found by NMR analysis to adopt a stable beta-hairpin structure in solution. Optimized peptides with micromolar receptor affinity exhibited high stability in biological fluids and inhibited cellular histamine release in an in vitro bioassay of IgE activity. The structure-activity relationships of these peptides, which are less than 1% of the size of IgE, suggest an overlap between their binding site and that of IgE on FcepsilonRI. Thus, the peptides demonstrate that blocking a small epitope on this receptor chain is sufficient to block IgE activity. Such structured peptides represent a possible starting point for the design of novel antagonists, and offer the potential for testing in vivo a new approach for treating allergic disease.

The PYRIN domain: a member of the death domain-fold superfamily.

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.

A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins.

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.

Tryptophan zippers: stable, monomeric beta -hairpins.

A structural motif, the tryptophan zipper (trpzip), greatly stabilizes the beta-hairpin conformation in short peptides. Peptides (12 or 16 aa in length) with four different turn sequences are monomeric and fold cooperatively in water, as has been observed previously for some hairpin peptides. However, the folding free energies of the trpzips exceed substantially those of all previously reported beta-hairpins and even those of some larger designed proteins. NMR structures of three of the trpzip peptides reveal exceptionally well-defined beta-hairpin conformations stabilized by cross-strand pairs of indole rings. The trpzips are the smallest peptides to adopt an unique tertiary fold without requiring metal binding, unusual amino acids, or disulfide crosslinks.

ICEBERG: a novel inhibitor of interleukin-1beta generation.

ProIL-1beta is a proinflammatory cytokine that is proteolytically processed to its active form by caspase-1. Upon receipt of a proinflammatory stimulus, an upstream adaptor, RIP2, binds and oligomerizes caspase-1 zymogen, promoting its autoactivation. ICEBERG is a novel protein that inhibits generation of IL-1beta by interacting with caspase-1 and preventing its association with RIP2. ICEBERG is induced by proinflammatory stimuli, suggesting that it may be part of a negative feedback loop. Consistent with this, enforced retroviral expression of ICEBERG inhibits lipopolysaccharide-induced IL-1beta generation. The structure of ICEBERG reveals it to be a member of the death-domain-fold superfamily. The distribution of surface charge is complementary to the homologous prodomain of caspase-1, suggesting that charge-charge interactions mediate binding of ICEBERG to the prodomain of caspase-1.

Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis.

Eglin c from the leech Hirudo medicinalis is a potent protein inhibitor of many serine proteinases including chymotrypsin and subtilisins. Unlike most small protein inhibitors whose solvent-exposed enzyme-binding loop is stabilized primarily by disulfide bridges flanking the reactive-site peptide bond, eglin c possesses an enzyme-binding loop supported predominantly by extensive electrostatic/H-bonding interactions involving three Arg residues (Arg48, Arg51, and Arg53) projecting from the scaffold of the inhibitor. As an adjacent residue, the C-terminal Gly70 participates in these interactions via its alpha-carboxyl group interacting with the side chain of Arg51 and the main chain of Arg48. In addition, the amide NH group of Gly70 donates an H-bond to the carbonyl C=O groups of Arg48 and Arg51. To understand the structural and functional relevance of the electrostatic/H-bonding network, we chemically synthesized wild-type eglin c and three analogues in which Gly70 was either deleted or replaced by glycine amide (NH(2)CH(2)CONH(2)) or by alpha-hydroxylacetamide (HOCH(2)CONH(2)). NMR analysis indicated that the core structure of eglin c was maintained in the analogues, but that the binding loop was significantly perturbed. It was found that deletion or replacement of Gly70 destabilized eglin c by an average of 2.7 kcal/mol or 20 degrees C in melting temperature. As a result, these inhibitors become substrates for their target enzymes. Binding assays on these analogues with a catalytically incompetent subtilisin BPN' mutant indicated that loss or weakening of the interactions involving the carboxylate of Gly70 caused a decrease in binding by approximately 2 orders of magnitude. Notably, for all four synthetic inhibitors, the relative free energy changes (DeltaDeltaG) associated with protein destabilization are strongly correlated (slope = 0.94, r(2) = 0. 9996) with the DeltaDeltaG values derived from a decreased binding to the enzyme.

Characterization of the binding interface between the E-domain of Staphylococcal protein A and an antibody Fv-fragment.

Staphylococcal protein A (SpA) is a cell-surface component of Staphylococcus aureus. In addition to the well-characterized interaction between SpA and the Fc-region of human IgG, an alternative binding interaction between SpA and the Fab-region of immunoglobulin domains encoded by the V(H)3 gene family has been described. To characterize structurally the interface formed by SpA repeats and type-3 V(H)-domains, we have studied the 32-kDa complex formed between an E-domain mutant (EZ4) and the Fv-fragment of the humanized anti-HER2 antibody (Hu4D5-8) using heteronuclear NMR spectroscopy. Protocols were established for efficient incorporation of (15)N, (13)C, and (2)H into EZ4 and the V(H)- and V(L)-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 and the V(H)-domain in both free and complexed states. Broadening of certain V(H)-resonances in the free and bound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencing significant chemical shift changes of backbone (1)H(N), (15)N, and (13)CO resonances upon complex formation delineate contiguous surfaces on EZ4 and the V(H)-domain that define the binding interfaces of the two proteins. The interaction surfaces identified by chemical shift mapping are comprised of predominantly hydrophilic residues. This is in contrast to the SpA-Fc interface which is predominantly hydrophobic in nature. Further analysis of the surface properties suggests a probable binding orientation for SpA- and V(H)3-domains.

Solution structure of the VEGF-binding domain of Flt-1: comparison of its free and bound states.

The extracellular portion of the VEGF and PlGF receptor, Flt-1 (or VEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1D2) is necessary and sufficient for high affinity VEGF binding. The 1.7 A resolution crystal structure of Flt-1D2 bound to VEGF revealed that this domain is a member of the I-set of the immunoglobulin superfamily, but has several unusual features including a region near the N terminus that bulges away from the domain rather than pairing with the neighboring beta-strand. Some of the residues in this region make contact with VEGF, raising the possibility that this bulge could be a consequence of VEGF binding and might not be present in the absence of ligand. Here we report the three-dimensional structure of Flt-1D2 in its uncomplexed form determined by NMR spectroscopy. A semi-automated method for NOE assignment that takes advantage of the previously solved crystal structure was used to facilitate rapid analysis of the 3D NOESY spectra. The solution structure is very similar to the previously reported VEGF-bound crystal structure; the N-terminal bulge is present, albeit in a different conformation. We also report the 2.7 A crystal structure of Flt-1D2 in complex with VEGF solved in a different crystal form that reveals yet another conformation for the N-terminal bulge region. (1)H-(15)N heteronuclear NOEs indicate this region is flexible in solution; the crystal structures show that this region is able to adopt more than one conformation even when bound to VEGF. Thus, VEGF-binding is not accompanied by significant structural change in Flt-1D2, and the unusual structural features of Flt-1D2 are an intrinsic property of this domain.

Antibody variable region binding by Staphylococcal protein A: thermodynamic analysis and location of the Fv binding site on E-domain.

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (V(H)), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3-4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5+/-0.5 x 10(5) M(-1). A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0+/-0.1, Ka = 2.0+/-0.3 x 10(5) M(-1), and deltaH = -7.1+/-0.4 kcal mol(-1). Similar binding thermodynamics are obtained for titration of the isolated V(H) domain with E-domain indicating that the E-domain binding site on Fab resides within V(H). E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2+/-0.1, Ka > 1.0 x 10(7) M(-1), and deltaH = -24.6+/-0.6 kcal mol(-1). Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the V(H) domain implicated previously in protein A binding.

The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII.

The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 0. 6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVII with TF. Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity.

Total chemical synthesis of bovine pancreatic trypsin inhibitor by native chemical ligation.

Bovine pancreatic trypsin inhibitor (BPTI) is an important model for the study of protein folding. Herein we describe a robust approach to the total chemical synthesis of BPTI using native chemical ligation of unprotected peptide segments in aqueous solution. After refolding and oxidative formation of disulfides, the target protein was purified by affinity chromatography. The synthetic BPTI was characterized by mass spectrometry, inhibition assay, thermal denaturation and 2D NMR spectroscopy, and was shown to be structurally and functionally identical to natural BPTI. The synthetic strategy presented in this paper has enabled us to establish rapid access to novel analogues of BPTI.

Solution structure of the heparin-binding domain of vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and is a potent angiogenic and vascular permeabilizing factor. VEGF is also an important mediator of pathological angiogenesis associated with cancer, rheumatoid arthritis and proliferative retinopathy. The binding of VEGF to its two known receptors, KDR and Flt-1, is modulated by cell-surface-associated heparin-like glycosaminoglycans and exogenous heparin or heparan sulfate. Heparin binding to VEGF165, the most abundantly expressed isoform of VEGF, has been localized to the carboxy-terminal 55 residues; plasmin cleavage of VEGF165 yields a homodimeric 110-residue amino-terminal receptor-binding domain (VEGF110) and two 55-residue carboxy-terminal heparin-binding fragments. The endothelial cell mitogenic potency of VEGF110 is decreased significantly relative to VEGF165, indicating that the heparin-binding domains are critical for stimulating endothelial cell proliferation. RESULTS: The solution structure of the 55-residue heparin-binding domain of VEGF165 has been solved using data from two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. The structure has two subdomains, each containing two disulfide bridges and a short two-stranded antiparallel beta sheet; the carboxy-terminal subdomain also contains a short alpha helix. Hydrophobic interactions are limited to sidechains packing against the disulfide bridges. CONCLUSIONS: The heparin-binding domain of VEGF has no significant sequence or structural similarity to any known proteins and thus represents a novel heparin-binding domain. Most of the positively charged amino acid sidechains are localized on one side of the carboxy-terminal subdomain or on an adjacent disordered loop in the amino-terminal subdomain. The observed distribution of surface charges suggests that these residues constitute a heparin interaction site.

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].

Structural mimicry of a native protein by a minimized binding domain.

The affinity between molecules depends both on the nature and presentation of the contacts. Here, we observe coupling of functional and structural elements when a protein binding domain is evolved to a smaller functional mimic. Previously, a 38-residue form of the 59-residue B-domain of protein A, termed Z38, was selected by phage display. Z38 contains 13 mutations and binds IgG only 10-fold weaker than the native B-domain. We present the solution structure of Z38 and show that it adopts a tertiary structure remarkably similar to that observed for the first two helices of B-domain in the B-domain/Fc complex [Deisenhofer, J. (1981) Biochemistry 20, 2361-2370], although it is significantly less stable. Based on this structure, we have improved on Z38 by designing a 34-residue disulfide-bonded variant (Z34C) that has dramatically enhanced stability and binds IgG with 9-fold higher affinity. The improved stability of Z34C led to NMR spectra with much greater chemical shift dispersion, resulting in a more precisely determined structure. Z34C, like Z38, has a structure virtually identical to the equivalent region from native protein A domains. The well-defined hydrophobic core of Z34C reveals key structural features that have evolved in this small, functional domain. Thus, the stabilized two-helix peptide, about half the size and having one-third of the remaining residues altered, accurately mimics both the structure and function of the native domain.

Solution structure of the E-domain of staphylococcal protein A.

The E-domain of staphylococcal protein A is one of five homologous IgG-binding domains designated E, D, A, B, and C that comprise the extracellular portion of protein A. The E-domain binds tightly to Fc fragments of IgG and binds certain Fv fragments with micromolar affinity. To explore further the structural features of Fc binding by protein A, and as a first step in developing a structural understanding of E-domain/Fv complex formation, we have determined the solution structure of the uncomplexed E-domain using 2D homonuclear and heteronuclear NMR spectroscopy. Complete 1H and 15N resonance assignments were obtained, and the structure was determined from 383 NOE-derived distance restrains, 34 phi and 19 chi 1 dihedral angle restraints, and 54 restraints for 27 H-bonds. 3JH alpha-H beta coupling constants and long-range NOEs involving Phe11 indicate the side chain exists in more than one conformation with differing chi 1 values. NOE restraints that were incompatible with chi 1 = -60 degrees were removed from one set of structure calculations, and those incompatible with chi 1 = 180 degrees were removed from a second set to allow Phe11 to explore both rotamer wells. Thus, two sets of 20 final structures, having no distance or dihedral angle restraint violations greater than 0.12 A or 1.6 degrees, respectively, represent the solution structure of the E-domain. Backbone atomic rms differences with respect to the mean coordinates for each set of 20 structures for residues 8-53 averaged 0.41 +/- 0.06 and 0.35 +/- 0.06 A. No significant differences in the overall structure result from the different orientations of Phe11. The solution structure of the E-domain consists of three alpha-helices that pack together to form a compact helical bundle. A detailed comparison between the E-domain ensembles and the previously determined structure for the B-domain in complex with Fc indicates that only the 180 degrees chi 1 rotamer of Phe11 is competent for binding; the -60 degrees chi 1 rotamer must reorient to 180 degrees to create a cavity that is filled by Ile253 from the CH2 domain of Fc in the Fc-bound complex.

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae.

The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy. Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified. yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin. As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR. Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM. Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites. Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl). Positive cooperativity was observed among all sites. Ca2+ binding was also monitored indirectly by one-dimensional NMR. Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III). All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2). Thus, an interaction between the two domains of intact yCaM affects the behavior of site III. These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed.

Sequential assignment of the backbone nuclei (1H, 15N and 13C) of c-H-ras p21 (1-166).GDP using a novel 4D NMR strategy.

The c-H-ras p21 protein is the product of the human ras proto-oncogene, a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. These proteins play an important role in the control of cellular growth. We report here the sequential assignment of the backbone nuclei in a truncated form of the 21-kD gene product, using our recently proposed 4D NMR strategy (Boucher et al., 1992). These assignments are the first step towards a full investigation of the structure, dynamics and interactions of wild-type and oncogenic ras p21 using NMR spectroscopy. Some of the data were presented at the 33rd ENC held at Asilomar, California, U.S.A., in April 1992.