Helix Bundle Structure Research Articles

Crystal Structures of Fusion Cores from CCoV-HuPn-2018 and SADS-CoV.

Cross-species spillover to humans of coronaviruses (CoVs) from wildlife animal reservoirs poses marked and global threats to human and animal health. Recently, sporadic infection of canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) in hospitalized patients with pneumonia genetically related to canine and feline coronavirus were identified. In addition, swine acute diarrhea syndrome coronavirus (SADS-CoV) had the capability of broad tropism to cultured cells including from humans. Together, the transmission of Alphacoronaviruses that originated in wildlife to humans via intermediate hosts was responsible for the high-impact emerging zoonosis. Entry of CoV is mainly mediated by Spike and formation of a typical six helix bundle (6-HB) structure in the postfusion state of Spike is pivotal. Here, we present the complete fusion core structures of CCoV-HuPn-2018 and SADS-CoV from Alphacoronavirus at 2.10 and 2.59 Å, respectively. The overall structure of the CCoV-HuPn-2018 fusion core is similar to Alphacoronavirus like HCoV-229E, while SADS-CoV is analogous to Betacoronavirus like SARS-CoV-2. Collectively, we provide a structural basis for the development of pan-CoV small molecules and polypeptides based on the HR1-HR2 complex, concerning CCoV-HuPn-2018 and SADS-CoV.

Crystal structures of QseE and QseG: elements of a three-component system from Escherichia coli.

Bacteria regulate virulence by using two-component systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). TCSs respond to environmental signals and change gene-expression levels. The HK QseE and the RR QseF regulate the virulence of Enterobacteriaceae bacteria such as enterohemorrhagic Escherichia coli. The operon encoding QseE/QseF also contains a gene encoding an outer membrane lipoprotein, qseG. The protein product QseG interacts with QseE in the periplasmic space to control the activity of QseE and constitutes a unique QseE/F/G three-component system. However, the structural bases of their functions are unknown. Here, crystal structures of the periplasmic regions of QseE and QseG were determined with the help of AlphaFold models. The periplasmic region of QseE has a helix-bundle structure as found in some HKs. The QseG structure is composed of an N-terminal globular domain and a long C-terminal helix forming a coiled-coil-like structure that contributes to dimerization. Comparison of QseG structures obtained from several crystallization conditions shows that QseG has structural polymorphisms at the C-terminus of the coiled-coil structure, indicating that the C-terminus is flexible. The C-terminal flexibility is derived from conserved hydrophilic residues that reduce the hydrophobic interaction at the coiled-coil interface. Electrostatic surface analysis suggests that the C-terminal coiled-coil region can interact with QseE. The observed structural fluctuation of the C-terminus of QseG is probably important for interaction with QseE.

Role and structure of the small subunit forming heterodimers with laccase-like enzymes.

Unlike laccases sensu stricto, which are usually monomeric enzymes, laccase-like enzymes recently re-classified as Novel Laccases (NLACs) are characterized by the formation of heterodimers with small proteins (subunits) of unknown function. Here the NLAC from Pleurotus eryngii (PeNL) and a small protein selected from the fungal genome, that is homologous to reported POXA3 from Pleurotus ostreatus, were produced in Aspergillus oryzae separately or together. The two proteins interacted regardless of whether the small subunit was co-expressed or exogenously added to the enzyme. The stability and catalytic activity of PeNL was significantly enhanced in the presence of the small subunit. SEC-MALS analysis confirmed that the complex PeNL-ss is a heterodimer of 77.4 kDa. The crystallographic structure of the small protein expressed in Escherichia coli was solved at 1.6 Å resolution. This is the first structure elucidated of a small subunit of a NLAC. The helix bundle structure of the small subunit accommodates well with the enzyme model structure, including interactions with specific regions of NLACs and some amino acid residues of the substrate-binding loops. This article is protected by copyright. All rights reserved.

Conformational changes of α-helical peptides with different hydrophobic residues induced by metal-ion binding

We designed peptides that formed helix bundle structures upon binding of the metal-ions to His residues to form a stable hydrophobic core, in order to analyze the effects of Ala, Val, Ile, and Leu residues, located in the hydrophobic core, together with His, on the conformational changes in respective peptides designated as HA, HV, HI, and HL, respectively. Circular dichroism measurements showed that HV and HI changed from random coil to helix bundle structures upon Zn2+ binding, similar to that observed for HA, while HL existed in the helix bundle structure even in the absence of Zn2+. Electron spin resonance measurements showed that Cu2+ coordination of HI and HL was quite different from that of HA and HV, indicating that HA and HV fluctuated to a greater extent in the solution, despite that their apparent α-helical contents being similar to those of HI and HL. This was also supported by the results obtained from the analyses of thermal stabilities. The change in the structural fluctuation for each peptide upon Zn2+ binding was evaluated based on binding thermodynamics using isothermal titration calorimetry. The structural flexibility in the metal-ion-bound state was found to be in the order HA > HV > HI, and that in the metal-ion-unbound state was found to be greater for HI than that for HL.

Characterization of BDG8: An antibody-human IL-2 complex that selectively activates the effector arm of the immune system.

e15004 Background: Interleukin 2 is a 15.4 kDa type I cytokine of the four helix bundle structure. IL-2 signaling has two opposite effects, it can enhance immune response by activation of effector cells and regulate immune response by activation proliferation of regulatory T cells (Tregs). IL-2 mediates its effect by binding to two forms of IL-2 receptor: i) trimeric receptors made of IL-2Rα (CD25), IL-2Rβ (CD122), and a common IL-2Rγ (γc, CD132) chains or ii) a dimeric receptor of only the IL-2Rβ and IL-2Rγ subunits. In Tregs, activation of the trimeric receptor is associated with FoxP3 mediated transcription leading to the production of suppression factors. Tregs express higher levels of the αβγ trimeric IL-2 receptor than effector cells. However binding of IL-2 to the βγ dimer is associated with activation of effector cells expressing relatively high levels of the βγ dimer and express low levels of trimer. High dose IL-2 therapy is used in melanoma and metastatic renal cell carcinoma with response rates 10%-15%. While efficacious, this approach has several disadvantages: 1) IL-2 dependent adverse effects such as Vascular Leak Syndrome (VLS) excluding many patients from being considered 2) The short half-life of IL-2 requires frequent administration, resulting in repeated spikes in the level of circulating IL-2. 3) Administered IL-2 is not selective and enhances a non-desired activation of Tregs. Methods: Antibodies were computational designed to bind to a specific epitope on Il-2 allowing for the binding to the CD122/CD132 complex and exclude the CD122/CD132/CD25 complex. Results: Here we show that we specifically designed and tested a humanized antibody (BD8) that: i) binds tightly the human IL-2 (hIL-2) and ii) the antibody- hIL-2 complex preferentially to bind the IL-2Rβ sub-unit and blocks the IL-2Rα subunit. We show administering the BD8 Ab-IL-2 complex to C57BL/6 mice has a profound effect on activation of MP CD8+, NK and NKT effector cells with minimal effect on Tregs. In mice the antibody-hIL-2 complex serum half -life is much longer then the half-life of hIL-2. In the B16-F10 Melanoma model, BD8-hIL2 resulted in robust effect compared to hIL-2 alone. Conclusions: BD8 is a computationally designed antibody specifically binds to IL-2 inducing the activation of effector cells without stimulating Tregs and demonstrates efficacy in animal cancer models.

Cost-effective downstream processing of recombinantly produced pexiganan peptide and its antimicrobial activity

Antimicrobial peptides (AMPs) have significant potential as alternatives to classical antibiotics. However, AMPs are currently prepared using processes which are often laborious, expensive and of low-yield, thus hindering their research and application. Large-scale methods for production of AMPs using a cost-effective approach is urgently required. In this study, we report a scalable, chromatography-free downstream processing method for producing an antimicrobial peptide, pexiganan, using recombinant Escherichia coli (E. coli). The four helix bundle structure of the unique carrier protein DAMP4 was used to facilitate a simple and cheap purification process based on a selective thermochemical precipitation. Highly pure fusion protein DAMP4var-pexiganan was obtained at high yield (around 24 mg per 800 mL cell culture with a final cultivation OD600 ~ 2). The purification yield of DAMP4var-pexiganan protein is increased twofold with a 72.9% of the protein recovery in this study as compared to the previous purification processes (Dwyer in Chem Eng Sci 105:12–21, 2014). The antimicrobial peptide pexiganan was released and activated from the fusion protein by a simple acid-cleavage. Isoelectric precipitation was then applied to separate the pexiganan peptide from the DAMP4var protein carrier. The final yield of pure bio-produced pexiganan was 1.6 mg from 800 mL of bacterial cell culture (final cultivation OD600 ~ 2). The minimum bactericidal concentration (MBC) test demonstrated that the bio-produced pexiganan has the same antimicrobial activity as chemically synthesized counterpart. This novel downstream process provides a new strategy for simple and probable economic production of antimicrobial peptides.

First observation of metal ion-induced structural fluctuations of α-helical peptides by using diffracted X-ray tracking

In order to analyze protein structural dynamics, we designed simple model peptides whose structures changed from random-coil to helix-bundle structures by forming stable hydrophobic core in the presence of metal ions. The strategy involved destabilizing a de novo designed three helix-bundle protein by substituting the residues present in its hydrophobic core with histidine and small amino acids. The conformational changes of peptides induced upon binding of Zn2+ to histidine were analyzed using circular dichroism spectroscopy, which revealed peptides, HA and HG, to be good candidates for further analyses. The diffracted X-ray tracking experiments showed that the structural fluctuations of both HA and HG were suppressed upon binding of Zn2+. We succeeded in observing the differences in fluctuations of HA and HG in solution between random-coil like and helix-bundle structures. The metal-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry.

A Novel Prediction Method of Protein Structural Classes Based on Protein Super-Secondary Structure

At present, the feature extraction of protein sequences is the most basic issue to predict protein structural classes and is also the key problem to decide the quality of prediction. In order to predict protein structural classes accurately, we construct a 14-dimensional feature vector based on protein secondary and super-secondary structure information to reflect the content and spatial ordering of the given protein sequences. Among the vector, seven features about -helix bundle, hairpin motifs, Rossman folds, -plaits and other super-secondary structure information are first proposed in our paper. Experiments show that our method improves overall accuracy of lower similarity datasets 1189 and 640 by 0.9% - 3.8% and 0.5% - 4.2% respectively compared with other methods and has a competitive advantage for predicting proteins in and classes.

G Protein-Coupled Receptors - Targets for Fragment-based Drug Discovery.

As the considerable technical challenges involved with generating crystal structures of G (guanine nucleotide- binding) protein-coupled receptors (GPCRs) are starting to be successfully addressed, opportunities to apply fragment-based drug discovery (FBDD) to this class of target are becoming a reality. GPCRs represent a large and important family of drug targets with considerable clinical and commercial interest. While their general seven transmembrane helix bundle structures are amenable to therapeutic intervention with small molecules, to date successful drugs have primarily been discovered using traditional competitive or function-based screening. With advances in biophysical screening techniques such as Surface Plasmon Resonance (SPR) and Target-Immobilised NMR Screening (TINS), being matched to developments in molecular dynamics simulations, virtual screening and stabilisation of biologically relevant conformations of GPCRs, structure-based approaches using fragment starting points are beginning to be applied to the discovery of new generations of small molecules.

Cloning, homology modeling, and reaction mechanism analysis of a novel cis-epoxysuccinate hydrolase from Klebsiella sp.

The gene encoding a novel cis-epoxysuccinate hydrolase, which hydrolyzes cis-epoxysuccinate to L (+)-tartaric acid, was cloned from Klebsiella sp. BK-58 and expressed in Escherichia coli. The ORF was 825 bp encoding a mature protein of 274 amino acids with a molecular mass of 30.1 kDa. Multiple sequence alignment showed that the enzyme belonged to the haloacid dehalogenase-like super family. Homology modeling and site-directed mutagenesis were performed to investigate the structural characteristics of the enzyme. Its overall structure consisted of a core domain formed by six-stranded parallel β-sheets flanked by seven α-helices and a subdomain that had a four helix bundle structure. Residues D48, T52, R85, N165, K195, Y201, A219, H221, and D224 were catalytically important forming the active pocket between the two domains. An (18)O-labeling study suggested that the catalytic reaction of the enzyme proceeded through a two-step mechanism.

Human antimicrobial peptides and proteins.

As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to combat drug-resistant superbugs, fungi, viruses, parasites, or cancer. Alternatively, multiple factors (e.g., albumin, arginine, butyrate, calcium, cyclic AMP, isoleucine, short-chain fatty acids, UV B light, vitamin D, and zinc) are able to induce the expression of antimicrobial peptides, opening new avenues to the development of anti-infectious drugs.

Intensified expression and purification of a recombinant biosurfactant protein

Proteins and peptides are emerging as components for novel materials that are switchable in response to their environment, and have enhanced sustainability over traditional materials. Proteins and peptides are known to be surface active and are widely used to stabilise foams and emulsions. However, designed surfactant proteins and peptides are presently produced using non-industrial processes based, for example, on costly chromatographic approaches developed for biopharmaceuticals. Here we report an intensified chromatography-free process for protein and peptide surfactant manufacture, for a recently-reported helix-bundle class of biosurfactants. The helix bundle structure is shown to remain stable and soluble under temperature and salt conditions that disrupt cells and precipitate cellular proteins. This finding opens a process route which simply involves heating cells, in fermentation media, to high temperature (e.g. 95°C), leading to release of soluble biosurfactant with simultaneous precipitation of contaminants. This “bake-to-break and precipitate” (BPP) process allows recovery of purified biosurfactant through simple thermal treatment followed by solid–liquid separation. Experiments were conducted with the four-helix bundle protein DAMP4, which was expressed intracellularly at a level of 15mgL−1 OD600−1 in fully synthetic minimal media. Thermal treatment of 100mL of E. coli suspension at OD600=4 produced 4.8mg of functional protein surfactant at a yield of 84% following simple microfiltration. Further polishing by precipitation and filtration gave 2.4mg of highly-pure biosurfactant. This work demonstrates that co-considered molecular and intensified process design can be used to progress the development of new biological products into low-cost industrial sectors such as those based on surfactants.

Dual Role of an N-terminal Amyloidogenic Mutation in Apolipoprotein A-I: DESTABILIZATION OF HELIX BUNDLE AND ENHANCEMENT OF FIBRIL FORMATION

A number of naturally occurring mutations of apolipoprotein (apo) A-I, the major protein of HDL, are known to be associated with hereditary amyloidosis and atherosclerosis. Here, we examined the effects of the G26R point mutation in apoA-I (apoA-I(Iowa)) on the structure, stability, and aggregation propensity to form amyloid fibril of full-length apoA-I and the N-terminal fragment of apoA-I. Circular dichroism and fluorescence measurements demonstrated that the G26R mutation destabilizes the N-terminal helix bundle domain of full-length protein, leading to increased hydrophobic surface exposure, whereas it has no effect on the initial structure of the N-terminal 1-83 fragment, which is predominantly a random coil structure. Upon incubation for extended periods at neutral pH, the N-terminal 1-83 variants undergo a conformational change to β-sheet-rich structure with a great increase in thioflavin T fluorescence, whereas no structural change is observed in full-length proteins. Comparison of fibril-forming propensity among substituted mutants at Gly-26 position of 1-83 fragments demonstrated that the G26R mutation enhances the nucleation step of fibril formation, whereas G26K and G26E mutations have small or inhibiting effects on the formation of fibrils. These fibrils of the 1-83 variants have long and straight morphology as revealed by atomic force microscopy and exhibited significant toxicity with HEK293 cells. Our results indicate dual critical roles of the arginine residue at position 26 in apoA-I(Iowa): destabilization of the N-terminal helix bundle structure in full-length protein and enhancement of amyloid fibril formation by the N-terminal 1-83 fragment.

Membrane-induced Lever Arm Expansion Allows Myosin VI to Walk with Large and Variable Step Sizes

Myosin VI, the only known minus-ended actin filament-dependent motor, plays diverse cellular roles both as a processive motor and as a mechanical anchor. Although myosin VI has a short lever arm containing only one "IQ-motif" and a unique insertion for CaM binding, the motor walks with large and variable step sizes of ∼30-36 nm. Here, we show that the previously predicted coiled-coil domain immediately following the IQ-motifs (referred to as the lever arm extension (LAE)) adopts a stable monomeric, three-helix bundle fold in solution. Importantly, the LAE can undergo reversible, lipid membrane-dependent conformational changes. Upon exposure to lipid membranes, the LAE adopts a partially extended rod shape, and the removal of lipids from the LAE converts it back into the compact helix bundle structure. Molecular dynamics simulations indicate that lipid membrane binding may initiate unfolding and thereby trigger the LAE expansion. This reversible, lipid membrane-dependent expansion of the LAE provides a mechanistic base for myosin VI to walk with large and variable step sizes.

Solution Structural Characterization of Coiled-Coil Peptide–Polymer Side-Conjugates

Detailed structural characterization of protein-polymer conjugates and understanding of the interactions between covalently attached polymers and biomolecules will build a foundation to design and synthesize hybrid biomaterials. Conjugates based on simple protein structures are ideal model system to achieve these ends. Here we present a systematic structural study of coiled-coil peptide-poly(ethylene glycol) (PEG) side-conjugates in solution, using circular dichroism, dynamic light scattering, and small-angle X-ray scattering, to determine the conformation of conjugated PEG chains. The overall size and shape of side-conjugates were determined using a cylindrical form factor model. Detailed structural information of the covalently attached PEG chains was extracted using a newly developed model where each peptide-PEG conjugate was modeled as a Gaussian chain attached to a cylinder, which was further arranged in a bundle-like configuration of three or four cylinders. The peptide-polymer side-conjugates were found to retain helix bundle structure, with the polymers slightly compressed in comparison with the conformation of free polymers in solution. Such detailed structural characterization of the peptide-polymer conjugates, which elucidates the conformation of conjugated PEG around the peptide and assesses the effect of PEG on peptide structure, will contribute to the rational design of this new family of soft materials.

The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima.

The bacterial histidine autokinase CheA contains a histidine phosphotransfer (Hpt) domain that accepts a phosphate from the catalytic domain and donates the phosphate to either target response regulator protein, CheY or CheB. The Hpt domain forms a helix-bundle structure with a conserved four-helix bundle motif and a variable fifth helix. Observation of two nearly equally populated conformations in the crystal structure of a Hpt domain fragment of CheA from Thermotoga maritima containing only the first four helices suggests more mobility in a tightly packed helix bundle structure than previously thought. In order to examine how the structures of Hpt domain homologs may differ from each other particularly in the conformation of the last helix, and whether an alternative conformation exists in the intact Hpt domain in solution, we have solved a high-resolution, solution structure of the CheA Hpt from T. maritima and characterized the backbone dynamics of this protein. The structure contains a four-helix bundle characteristic of histidine phosphotransfer domains. The position and orientation of the fifth helix resembles those in known Hpt domain crystal and solution structures in other histidine kinases. The alternative conformation that was reported in the crystal structure of the CheA Hpt from T. maritima missing the fifth helix is not detected in the solution structure, suggesting a role for the fifth helix in providing stabilizing forces to the overall structure.

A Membrane Cell for On-line Hydrogen/Deuterium Exchange to Study Protein Folding and Protein-Protein Interactions by Mass Spectrometry

A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.

Effect of polarization on the stability of a helix dimer

Molecular dynamics (MD) simulations have been carried out to study helix–helix interaction using both standard AMBER and polarized force fields. Comparison of the two simulations shows that electrostatic polarization of intra-protein hydrogen bonds plays a significant role in stabilizing the structure of helix dimer. This stabilizing effect is clearly demonstrated by examining the monomer structure, helix crossing angle and stability of backbone hydrogen bonds under AMBER and PPC. Since reliable prediction of protein–protein structure is a significant challenge, the current study should help shed light on the importance of electrostatic polarization of protein in helix–helix interaction and helix bundle structures.

Influence of N-terminal helix bundle stability on the lipid-binding properties of human apolipoprotein A-I

As the principal component of high-density lipoprotein (HDL), apolipoprotein (apo) A-I plays essential roles in lipid transport and metabolism. Because of its intrinsic conformational plasticity and flexibility, the molecular details of the tertiary structure of lipid-free apoA-I have not been fully elucidated. Previously, we demonstrated that the stability of the N-terminal helix bundle structure is modulated by proline substitution at the most hydrophobic region (residues around Y18) in the N-terminal domain. Here we examine the effect of proline substitution at S55 located in another relatively hydrophobic region compared to most of the helix bundle domain to elucidate the influences on the helix bundle structure and lipid interaction. Fluorescence measurements revealed that the S55P mutation had a modest effect on the stability of the bundle structure, indicating that residues around S55 are not pivotally involved in the helix bundle formation, in contrast to the insertion of proline at position 18. Although truncation of the C-terminal domain (Δ190–243) diminishes the lipid binding of apoA-I molecule, the mutation S55P in addition to the C-terminal truncation (S55P/Δ190–243) restored the lipid binding, suggesting that the S55P mutation causes a partial unfolding of the helix bundle to facilitate lipid binding. Furthermore, additional proline substitution at Y18 (Y18P/S55P/Δ190–243), which leads to a drastic unfolding of the helix bundle structure, yielded a greater lipid binding ability. Thus, proline substitutions in the N-terminal domain of apoA-I that destabilized the helix bundle promoted lipid solubilization. These results suggest that not only the hydrophobic C-terminal helical domain but also the stability of the N-terminal helix bundle in apoA-I are important modulators of the spontaneous solubilization of membrane lipids by apoA-I, a process that leads to the generation of nascent HDL particles.

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram-negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix-rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N-terminal domain of Thermus thermophilus PilC. The N-domain forms a dimer, adopting a six helix bundle structure with an up-down-up-down-up-down topology. The monomers are related by a rotation of 170 degrees , followed by a translation along the axis of the final alpha-helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram-negative bacteria. Site-directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function.