Conformational specificity of the lac repressor coiled-coil tetramerization domain

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.     

A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins.

In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl. Activation of the Abl transforming function has been shown to require two distinct domains of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176 to 242). The amino acid sequence of domain 1 indicates that it may be a coiled-coil oligomerization domain. We show here that domain 1 of Bcr forms a homotetramer. Tetramerization of Bcr-Abl through Bcr domain 1 correlates with activation of the tyrosine kinase and F-actin-binding functions of Abl. Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function as well as the ability of Bcr-Abl to transform Rat-1 fibroblasts or to abrogate interleukin-3 dependence in lymphoid cells. These results strongly suggest that Bcr-Abl oligomers are the active entities in transformation.     

Predicting helix orientation for coiled-coil dimers

The alpha-helical coiled coil is a structurally simple protein oligomerization or interaction motif consisting of two or more alpha helices twisted into a supercoiled bundle. Coiled coils can differ in their stoichiometry, helix orientation, and axial alignment. Because of the near degeneracy of many of these variants, coiled coils pose a challenge to fold recognition methods for structure prediction. Whereas distinctions between some protein folds can be discriminated on the basis of hydrophobic/polar patterning or secondary structure propensities, the sequence differences that encode important details of coiled-coil structure can be subtle. This is emblematic of a larger problem in the field of protein structure and interaction prediction: that of establishing specificity between closely similar structures. We tested the behavior of different computational models on the problem of recognizing the correct orientation--parallel vs. antiparallel--of pairs of alpha helices that can form a dimeric coiled coil. For each of 131 examples of known structure, we constructed a large number of both parallel and antiparallel structural models and used these to assess the ability of five energy functions to recognize the correct fold. We also developed and tested three sequence-based approaches that make use of varying degrees of implicit structural information. The best structural methods performed similarly to the best sequence methods, correctly categorizing approximately 81% of dimers. Steric compatibility with the fold was important for some coiled coils we investigated. For many examples, the correct orientation was determined by smaller energy differences between parallel and antiparallel structures distributed over many residues and energy components. Prediction methods that used structure but incorporated varying approximations and assumptions showed quite different behaviors when used to investigate energetic contributions to orientation preference. Sequence based methods were sensitive to the choice of residue-pair interactions scored.     

Structure of the Bcr-Abl oncoprotein oligomerization domain.

The Bcr-Abl oncoprotein is responsible for a wide range of human leukemias, including most cases of Philadelphia chromosome-positive chronic myelogenous leukemia. Oligomerization of Bcr-Abl is essential for oncogenicity. We determined the crystal structure of the N-terminal oligomerization domain of Bcr-Abl (residues 1-72 or Bcr1-72) and found a novel mode of oligomer formation. Two N-shaped monomers dimerize by swapping N-terminal helices and by forming an antiparallel coiled coil between C-terminal helices. Two dimers then stack onto each other to form a tetramer. The Bcr1-72 structure provides a basis for the design of inhibitors of Bcr-Abl transforming activity by disrupting Bcr-Abl oligomerization.     

Multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone

The alpha-helical coiled coil can adopt a variety of topologies, among the most common of which are parallel and antiparallel dimers and trimers. We present Multicoil2, an algorithm that predicts both the location and oligomerization state (two versus three helices) of coiled coils in protein sequences. Multicoil2 combines the pairwise correlations of the previous Multicoil method with the flexibility of Hidden Markov Models (HMMs) in a Markov Random Field (MRF). The resulting algorithm integrates sequence features, including pairwise interactions, through multinomial logistic regression to devise an optimized scoring function for distinguishing dimer, trimer and non-coiled-coil oligomerization states; this scoring function is used to produce Markov Random Field potentials that incorporate pairwise correlations localized in sequence. Multicoil2 significantly improves both coiled-coil detection and dimer versus trimer state prediction over the original Multicoil algorithm retrained on a newly-constructed database of coiled-coil sequences. The new database, comprised of 2,105 sequences containing 124,088 residues, includes reliable structural annotations based on experimental data in the literature. Notably, the enhanced performance of Multicoil2 is evident when tested in stringent leave-family-out cross-validation on the new database, reflecting expected performance on challenging new prediction targets that have minimal sequence similarity to known coiled-coil families. The Multicoil2 program and training database are available for download from http://multicoil2.csail.mit.edu.     

Importance of potential interhelical salt-bridges involving interior residues for coiled-coil stability and quaternary structure

Coiled coils are formed by two or more alpha-helices that align in a parallel or an antiparallel relative orientation. Polar interactions involving residues at the interior a and d positions are important for determining the quaternary structure of coiled coils. In the model heterodimeric coiled-coil Acid-a1-Base-a1, a buried a-d' Asn-Asn interaction is sufficient to specify both a dimeric structure and an antiparallel relative helix orientation. Although the equivalent a-a' interaction is found in parallel coiled coils, there is no example of an a-d' Asn-Asn interaction in structurally characterized, naturally occurring antiparallel coiled coils. Instead, interior charged residues form interhelical salt-bridges with residues at the adjacent e or g positions. Using a model coiled-coil heterodimer, we have explored the role of a potential interhelical interaction between an Arg at an interior d position and a Glu at the adjacent g' position. Our results demonstrate that this potentially attractive interhelical Coulombic interaction has little or no influence on helix orientation. Instead, we show that burying a single Arg residue at an interior position is sufficient to specify a dimeric state at a significantly lower thermodynamic cost than burial of two interacting Asn residues.     

Orientational Ambiguity in Septin Coiled Coils and its Structural Basis

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function.     

Conformation of the Drosophila motor protein non-claret disjunctional in solution from X-ray and neutron scattering

The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5.     

How oligomerization contributes to the thermostability of an archaeon protein. Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii

To study how oligomerization may contribute to the thermostability of archaeon proteins, we focused on a hexameric protein, protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii (StoPIMT). The crystal structure shows that StoPIMT has a distinctive hexameric structure composed of monomers consisting of two domains: an S-adenosylmethionine-dependent methyltransferase fold domain and a C-terminal alpha-helical domain. The hexameric structure includes three interfacial contact regions: major, minor, and coiled-coil. Several C-terminal deletion mutants were constructed and characterized. The hexameric structure and thermostability were retained when the C-terminal alpha-helical domain (Tyr(206)-Thr(231)) was deleted, suggesting that oligomerization via coiled-coil association using the C-terminal alpha-helical domains did not contribute critically to hexamerization or to the increased thermostability of the protein. Deletion of three additional residues located in the major contact region, Tyr(203)-Asp(204)-Asp(205), led to a significant decrease in hexamer stability and chemico/thermostability. Although replacement of Thr(146) and Asp(204), which form two hydrogen bonds in the interface in the major contact region, with Ala did not affect hexamer formation, these mutations led to a significant decrease in thermostability, suggesting that two residues in the major contact region make significant contributions to the increase in stability of the protein via hexamerization. These results suggest that cooperative hexamerization occurs via interactions of "hot spot" residues and that a couple of interfacial hot spot residues are responsible for enhancing thermostability via oligomerization.     

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer–dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer–dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.     

Autoinhibition of Bcr-Abl through its SH3 domain

Bcr-Abl is a dysregulated tyrosine kinase whose mechanism of activation is unclear. Here, we demonstrate that, like c-Abl, Bcr-Abl is negatively regulated through its SH3 domain. Kinase activity, transformation, and leukemogenesis by Bcr-Abl are greatly impaired by mutations of the Bcr coiled-coil domain that disrupt oligomerization, but restored by an SH3 point mutation that blocks ligand binding or a complementary mutation at the intramolecular SH3 binding site defined in c-Abl. Phosphorylation of tyrosines in the activation loop of the catalytic domain and the linker between the SH2 and catalytic domains (SH2-CD linker) is dependent on oligomerization and required for leukemogenesis. These results suggest that Bcr-Abl has a monomeric, unphosphorylated state with the SH3 domain engaged intramolecularly to Pro1124 in the SH2-CD linker, the form that is sensitive to the inhibitor imatinib (STI-571). The sole function of the coiled-coil domain is to disrupt the autoinhibited conformation through oligomerization and intermolecular autophosphorylation.     

Mapping of structural determinants for the oligomerization of p58, a lectin-like protein of the intermediate compartment and cis-Golgi.

Shortly after synthesis, p58, the rat homologue of the mannose-binding lectin ERGIC-53/MR60, which localizes to pre-Golgi and cis-Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane-proximal half of the lumenal domain consists of four predicted alpha-helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled-coil interactions. Using single-stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the alpha-helical domains was probed by internal deletions. The N-terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild-type protein. The two membrane-proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual alpha-helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to D-mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild-type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58.     

The use of coiled-coil interactions for the analysis and micropreparative isolation of adenomatous polyposis coli protein complexes.

The coiled coil is a common structural motif found both as the dominant structure in fibrous proteins and as an oligomerization domain in a variety of cytoskeletal and extracellular matrix proteins. Coiled-coils typically consist of two to four helices that are supercoiled around one another in either parallel or antiparallel orientations. In the past few years our knowledge of the structure and specificity of coiled coil interactions has increased, allowing the de novo design and preparation of coiled-coils with well-defined structure and specificity. Indeed, the design and synthesis of a peptide that binds specifically to a single coiled-coil-containing protein, adenomatous polyposis coli (APC) has been reported. We have optimized solid-phase synthesis techniques to produce a modified form of the anti-APC peptide that contains a biotin moiety specifically placed so as to allow selective orientation onto the surface of a biosensor or affinity support. These peptide surfaces have been used to both monitor and purify APC and APC complexes from cellular extracts.     

Evaluation of the energetic contribution of interhelical Coulombic interactions for coiled coil helix orientation specificity.

Coiled coils are formed by two or more alpha-helices that align in a parallel or an antiparallel relative orientation. The factors that determine a preference for a given relative helix orientation are incompletely understood. The helix orientation preference for the designed coiled coil, Acid-a1-Base-a1, was measured previously. This model system therefore provides a means for the experimental determination of the energetic contribution of a variety of interactions to helix orientation specificity.The antiparallel preference for Acid-a1-Base-a1 is imparted by a single buried polar interaction. Interhelical Coulombic interactions between residues at the e and g positions have been proposed to influence helix orientation preference. In the Acid-a1-Base-a1 heterodimer, potentially attractive Coulombic interactions are expected in both orientations. To determine the energetic consequences of Coulombic interactions for helix orientation preference, we have positioned a single charged residue in each peptide such that exclusively favorable interhelical Coulombic interactions can occur only in the parallel orientation. In contrast, two potentially repulsive interactions are expected in the antiparallel orientation. Because the buried polar interaction can occur only in the antiparallel orientation, interhelical Coulombic interactions favor the parallel orientation and the potential to form a buried polar interaction favors the antiparallel orientation. We find no clear preference for an antiparallel orientation in the resulting heterodimer, Acid-Ke-Base-Eg, suggesting that interhelical Coulombic interactions and a buried polar interaction are of approximately equal importance for helix orientation specificity. Stability measurements indicate that maintenance of all favorable electrostatic interactions and/or avoidance of two potentially repulsive interactions contributes approximately 2.1 kcal/mol to helix orientation preference.     

Core residue replacements cause coiled-coil orientation switching in vitro and in vivo: structure-function correlations for osmosensory transporter ProP

Protein ProP acts as an osmosensory transporter in diverse bacteria. C-Terminal residues 468-497 of Escherichia coli ProP (ProPEc) form a four-heptad homodimeric alpha-helical coiled coil. Arg 488, at a core heptad a position, causes it to assume an antiparallel orientation. Arg in the hydrophobic core of coiled coils is destabilizing, but Arg 488 forms stabilizing interstrand salt bridges with Asp 475 and Asp 478. Mutation R488I destabilizes the coiled coil and elevates the osmotic pressure at which ProPEc activates. It may switch the coiled-coil orientation to parallel by eliminating the salt bridges and increasing the hydrophobicity of the core. In this study, mutations D475A and D478A, which disrupt the salt bridges without increasing the hydrophobicity of the coiled-coil core, had the expected modest impacts on the osmotic activation of ProPEc. The five-heptad coiled coil of Agrobacterium tumefaciens ProP (ProPAt) has K498 and R505 at a positions. Mutation K498I had little effect on the osmotic activation of ProPAt, and ProPAt-R505I was activated only at high osmotic pressure; on the other hand, the double mutant was refractory to osmotic activation. Both a synthetic peptide corresponding to ProPAt residues 478-516 and its K498I variant maintained the antiparallel orientation. The single R505I substitution created an unstable coiled coil with little orientation preference. Double mutation K498I/R505I switched the alignment, creating a stable parallel coiled coil. In vivo cross-linking showed that the C-termini of ProPAt and ProPAt-K498I/R505I were antiparallel and parallel, respectively. Thus, the antiparallel orientation of the ProP coiled coil is contingent on Arg in the hydrophobic core and interchain salt bridges. Two key amino acid replacements can convert it to a stable parallel structure, in vitro and in vivo. An intermolecular antiparallel coiled coil, present on only some orthologues, lowers the osmotic pressure required to activate ProP. Formation of a parallel coiled coil renders ProP inactive.     

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm.     

Regulation of c-Fes tyrosine kinase and biological activities by N-terminal coiled-coil oligomerization domains

The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains.     

Crystal structure of the coiled-coil dimerization motif of geminin: structural and functional insights on DNA replication regulation

We have determined the crystal structure of the coiled-coil domain of human geminin, a DNA synthesis inhibitor in higher eukaryotes. We show that a peptide encompassing the five heptad repeats of the geminin leucine zipper (LZ) domain is a dimeric parallel coiled coil characterized by a unique pattern of internal polar residues and a negatively charged surface that may target the basic domain of interacting partners. We show that the LZ domain itself is not sufficient to inhibit DNA synthesis but upstream and downstream residues are required. Analysis of a functional form of geminin by density sedimentation indicates an oligomeric structure. X-ray solution scattering experiments performed on a non-functional form of geminin having upstream basic residues and the LZ domain show a tetramer structure. Altogether, these results give a consistent identification and mapping of geminin interacting regions onto structurally important domains. They also suggest that oligomerization properties of geminin may be implicated in its inhibitory activity of DNA synthesis.     

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.