Four-helix Bundle Research Articles

Phylogenetic analysis of Harmonin homology domains

BackgroundHarmonin Homogy Domains (HHD) are recently identified orphan domains of about 70 residues folded in a compact five alpha-helix bundle that proved to be versatile in terms of function, allowing for direct binding to a partner as well as regulating the affinity and specificity of adjacent domains for their own targets. Adding their small size and rather simple fold, HHDs appear as convenient modules to regulate protein–protein interactions in various biological contexts. Surprisingly, only nine HHDs have been detected in six proteins, mainly expressed in sensory neurons.ResultsHere, we built a profile Hidden Markov Model to screen the entire UniProtKB for new HHD-containing proteins. Every hit was manually annotated, using a clustering approach, confirming that only a few proteins contain HHDs. We report the phylogenetic coverage of each protein and build a phylogenetic tree to trace the evolution of HHDs. We suggest that a HHD ancestor is shared with Paired Amphipathic Helices (PAH) domains, a four-helix bundle partially sharing fold and functional properties. We characterized amino-acid sequences of the various HHDs using pairwise BLASTP scoring coupled with community clustering and manually assessed sequence features among each individual family. These sequence features were analyzed using reported structures as well as homology models to highlight structural motifs underlying HHDs fold. We show that functional divergence is carried out by subtle differences in sequences that automatized approaches failed to detect.ConclusionsWe provide the first HHD databases, including sequences and conservation, phylogenic trees and a list of HHD variants found in the auditory system, which are available for the community. This case study highlights surprising phylogenetic properties found in orphan domains and will assist further studies of HHDs. We unveil the implication of HHDs in their various binding interfaces using conservation across families and a new protein–protein surface predictor. Finally, we discussed the functional consequences of three identified pathogenic HHD variants involved in Hoyeraal-Hreidarsson syndrome and of three newly reported pathogenic variants identified in patients suffering from Usher Syndrome.

The Ion-Translocating NrfD-Like Subunit of Energy-Transducing Membrane Complexes.

Several energy-transducing microbial enzymes have their peripheral subunits connected to the membrane through an integral membrane protein, that interacts with quinones but does not have redox cofactors, the so-called NrfD-like subunit. The periplasmic nitrite reductase (NrfABCD) was the first complex recognized to have a membrane subunit with these characteristics and consequently provided the family's name: NrfD. Sequence analyses indicate that NrfD homologs are present in many diverse enzymes, such as polysulfide reductase (PsrABC), respiratory alternative complex III (ACIII), dimethyl sulfoxide (DMSO) reductase (DmsABC), tetrathionate reductase (TtrABC), sulfur reductase complex (SreABC), sulfite dehydrogenase (SoeABC), quinone reductase complex (QrcABCD), nine-heme cytochrome complex (NhcABCD), group-2 [NiFe] hydrogenase (Hyd-2), dissimilatory sulfite-reductase complex (DsrMKJOP), arsenate reductase (ArrC) and multiheme cytochrome c sulfite reductase (MccACD). The molecular structure of ACIII subunit C (ActC) and Psr subunit C (PsrC), NrfD-like subunits, revealed the existence of ion-conducting pathways. We performed thorough primary structural analyses and built structural models of the NrfD-like subunits. We observed that all these subunits are constituted by two structural repeats composed of four-helix bundles, possibly harboring ion-conducting pathways and containing a quinone/quinol binding site. NrfD-like subunits may be the ion-pumping module of several enzymes. Our data impact on the discussion of functional implications of the NrfD-like subunit-containing complexes, namely in their ability to transduce energy.

Structural insights into the ligand recognition and catalysis of the key aminobutanoyltransferase CntL in staphylopine biosynthesis

Staphylopine (StP) and other nicotianamine-like metallophores are crucial for many pathogens to acquire the transition metals from hosts during invasion. CntL from Staphylococcus aureus (SaCntL) catalyzes the condensation of the 2-aminobutyrate (Ab) moiety of S-adenosylmethionine (SAM) with D-histidine in the biosynthesis of StP. Here, we report the crystal structures of SaCntL in complex with either SAM or two products. The structure of SaCntL consists of an N-terminal four-helix bundle (holding catalytic residue E84) and a C-terminal Rossmann fold (binding the substrates). The sequence connecting the N- and C-terminal domains (N-C linker) in SaCntL was found to undergo conformational alternation between open and closed states. Our structural and biochemical analyses suggested that this intrinsically dynamic interdomain linker forms an additional structural module that plays essential roles in ligand diffusion, recognition, and catalysis. We confirmed that SaCntL stereoselectively carries out the catalysis of D-His but not its enantiomer, L-His, and we found that the N-C linker and active site of SaCntL could accommodate both enantiomers. SaCntL is likely able to bind L-His without catalysis, and as a result, L-His could show inhibitory effects toward SaCntL. These findings provide critical structural and mechanistic insights into CntL, which facilitates a better understanding of the biosynthesis of nicotianamine-like metallophores and the discovery of inhibitors of this process.

The LDL receptor binding domain of apolipoprotein E directs the relative orientation of its C-terminal segment in reconstituted nascent HDL

Apolipoprotein E (apoE) (299 residues) is a highly helical protein that plays a critical role in cholesterol homeostasis. It comprises a four-helix bundle N-terminal (NT) and a C-terminal (CT) domain that can exist in lipid-free and lipid-associated states. In humans, there are two major apoE isoforms, apoE3 and apoE4, which differ in a single residue in the NT domain, with apoE4 strongly increasing risk of Alzheimer's disease (AD) and cardiovascular diseases (CVD). It has been proposed that the CT domain initiates rapid lipid binding, followed by a slower NT domain helix bundle opening and lipid binding to yield discoidal reconstituted high density lipoprotein (rHDL). However, the contribution of the NT domain on the CT domain organization in HDL remains poorly understood. To understand this, we employed Cys-specific cross-linking and spatially-sensitive fluorophores in the NT and CT domains of apoE3 and apoE4, and in isolated CT domain. We noted that the helices in isolated CT domain are oriented parallel to those in the neighboring molecule in rHDL, whereas full length apoE3 and apoE4 adopt either an anti-parallel or hairpin-like organization. It appears that the bulky NT domain determines the spatial organization of its CT domain in rHDL, a finding that has significance for apoE4, which is more susceptible to proteolytic cleavage in AD brains, showing increased accumulation of neurotoxic NT and CT fragments. We envisage that the structural organization of HDL apoE would have profound functional consequences in its ability to regulate cholesterol homeostasis in AD and CVD.

Rational thermostabilisation of four-helix bundle dimeric de novo proteins

The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

Salivary complement inhibitors from mosquitoes: Structure and mechanism of action

Inhibition of the alternative pathway (AP) of complement by saliva from Anopheles mosquitoes facilitates feeding by blocking production of the anaphylatoxins C3a and C5a, which activate mast cells leading to plasma extravasation, pain, and itching. We have previously shown that albicin, a member of the SG7 protein family from An. Albimanus, blocks the AP by binding to and inhibiting the function of the C3 convertase, C3bBb. Here we show that SG7.AF, the albicin homolog from An. freeborni, has a similar potency to albicin but is more active in the presence of properdin, a plasma protein that acts to stabilize C3bBb. Conversely, albicin is highly active in the absence or presence of properdin. Albicin and SG7.AF stabilize the C3bBb complex in a form that accumulates on surface plasmon resonance (SPR) surfaces coated with properdin, but SG7.AF binds with lower affinity than albicin. Albicin induces oligomerization of the complex in solution, suggesting that it is oligomerization that leads to stabilization on SPR surfaces. Anophensin, the albicin ortholog from An. stephensi, is only weakly active as an inhibitor of the AP, suggesting that the SG7 family may play a different functional role in this species and other species of the subgenus Cellia, containing the major malaria vectors in Africa and Asia. Crystal structures of albicin and SG7.AF reveal a novel four-helix bundle arrangement that is stabilized by an N-terminal hydrogen bonding network. These structures provide insight into the SG7 family and related mosquito salivary proteins including the platelet-inhibitory 30 kDa family.

Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA

The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1–independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein–lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain–binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity.

The Fusion Between Autophagic Vesicles and Lysosomes.

The autophagosome delivers engulfed substrates to the lysosome for degradation via membrane fusion between the autophagosome and the lysosome. The process of membrane fusion is highly conserved in evolution. It is widely accepted that membrane fusion in general is driven by the zippering of the SNARE complex to form a four-helix bundle. Besides SNAREs, other proteins are required to complete fusion efficiently, including tethering proteins, Rab GTPases, and SM proteins (Sec1/SM family proteins). This chapter will summarize the current knowledge of the key machinery involved in autophagosome-lysosome fusion, including autophagic SNAREs, involved ATG proteins, the HOPS complex, Rab GTPase, and other relevant aspects.

Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis.

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.

Allosteric cooperation in a de novo-designed two-domain protein

We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis-Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter Km and a 7-fold decrease in kcat These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.

Protein Residues and a Novel Motif Involved in the Cellular Localization of CheZ in Azorhizobium caulinodans ORS571.

Chemotaxis is essential for the competitiveness of motile bacteria in complex and harsh environments. The localization of chemotactic proteins in the cell is critical for coordinating a maximal response to chemotactic signals. One chemotaxis protein with a well-defined subcellular localization is the phosphatase CheZ. CheZ localizes to cell poles by binding with CheA in Escherichia coli and other enteric bacteria, or binding with a poorly understood protein called ChePep in epsilon-Proteobacteria. In alpha-Proteobacteria, CheZ lacks CheA-binding sites, and its cellular localization remains unknown. We therefore determined the localization of CheZ in the alpha-Proteobacteria Azorhizobium caulinodans ORS571. A. caulinodans CheZ, also termed as CheZAC, was found to be located to cell poles independently of CheA, and we suspect that either the N-terminal helix or the four-helix bundle of CheZAC is sufficient to locate to cell poles. We also found a novel motif, AXXFQ, which is adjacent to the phosphatase active motif DXXXQ, which effects the monopolar localization of CheZAC. This novel motif consisting of AXXFQ is conserved in CheZ and widely distributed among Proteobacteria. Finally, we found that the substitution of phosphatase active site affects the polar localization of CheZAC. In total, this work characterized the localization pattern of CheZ containing a novel motif, and we mapped the regions of CheZAC that are critical for its polar localization.

Effects of Excipients on the Structure and Dynamics of Filgrastim Monitored by Thermal Unfolding Studies by CD and NMR Spectroscopy.

Product excipients are used to confer a number of desirable properties on the drug substance to maintain or improve stability and facilitate drug delivery. This is especially important for products where the active pharmaceutical ingredient (API) is a recombinant protein. In this study, we aimed to determine if excipients and formulation conditions affect the structure and/or modulate the dynamics of the protein API of filgrastim products. Samples of uniformly labeled 15N-Met-granulocyte-colony stimulating factor (GCSF) were prepared at 100 μM (near formulation concentration) with various concentrations of individual components (polysorbate-20 and -80, sorbitol) and three pH values. Nuclear magnetic resonance (NMR) spectroscopy techniques were applied to measure chemical shift perturbation (CSP) to detect structural changes, and relaxation parameters (T1, T2, and heteronuclear Overhauser effect) were measured to probe the effects on protein backbone motions. In parallel, the same solution conditions were subjected to protein thermal unfolding studies monitored by circular dichroism spectropolarimetry (CD). Detergents (polysorbate-20 and 80) do not induce any observable changes on the protein structure and do not modify its dynamics at formulation concentration. Lowering pH to 4.0, a condition known to stabilize the conformation of filgrastim, as well as the addition of sorbitol produced changes of the fast motion dynamics in the nanosecond and picosecond timescale. NMR-derived order parameters, which measure the local conformational entropy of the protein backbone, show that lowering pH leads to a compaction of the four-helix bundle while the addition of sorbitol relaxes helices B and C, thereby reducing the mobility of loop CD. CSPs and measurements of protein dynamics via NMR-derived order parameters provide a description in structural and motional terms at an atomic resolution on how formulation components contribute to the stabilization of filgrastim products.

M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end

Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin-tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T's N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin-tropomyosin binding and weaker tropomyosin-actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin-thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM.

The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.

An angular motion of a conserved four-helix bundle facilitates alternating access transport in the TtNapA and EcNhaA transporters

There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, respectively. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using cross-linking experiments.

De Novo Design of Granulopoietic Proteins

Protein therapeutics are clinically developed and used as minorly engineered forms of their natural templates. This direct adoption of natural proteins in therapeutic contexts very frequently faces major challenges, including instability, poor solubility, and aggregation, which may result in undesired clinical outcomes. In contrast to classical protein engineering techniques, de novo protein design enables the introduction of radical sequence and structure manipulations, which can be used to address these challenges. In this work, we test the utility of two different design strategies to design novel granulopoietic proteins, using structural information from human granulocyte-colony stimulating factor (hG-CSF) as a template. The two strategies are: (1) An epitope rescaffolding where we migrate a tertiary structural epitope to simpler, idealised, proteins scaffolds (Fig. 1A-C), and (2) a topological refactoring strategy, where we change the protein fold by rearranging connections across the secondary structures and optimised the designed sequence of the new fold (Fig. 1A,D,E). Testing only eight designs, we obtained novel granulopoietic proteins that bind to the G-CSF receptor, have nanomolar activity in cell-based assays, and were highly thermostable and protease-resistant. NMR structure determination showed three designs to match their designed coordinates within less than 2.5 Å. While the designs possessed starkly different sequence and structure from the native G-CSF, they showed very specific activity in differentiating primary human haematopoietic stem cells into fully mature granulocytes. Morever, one design shows significant and specific activity in vivo in zebrafish and mice. These results are prospectively directing us to investigate the role of dimerisation geometry of G-GCSF receptor on activation magnitude and downstream signalling pathways. More broadly, the results also motivate our ongoing work on to design other heamatopoietic agents. In conclusion, our findings highlight the utility of computational protein design as a highly effective and guided means for discovering nover receptor modulators, and to obtain new mechanistic information about the target molecule. Figure 1. Two different strategies to generate superfolding G-CSF designs. (A) X-ray structure of G-CSF (orange) bound to its cognate receptor (red) through its binding epitope (blue). According to the epitope rescaffolding strategy, (B) the critical binding epitope residues were disembodied and used as a geometric search query against the entire Protein Data Bank (PDB) to retrieve structurally compatible scaffolds. The top six compatible scaffolds structures are shown in cartoon representation. (C) The top two templates chosen for sequence design, were a de novo designed coiled-coil and a four-helix bundle with unknown function. The binding epitopes were grafted, and the scaffolds were optimised to rigidly host the guest epitope. (D-E) According to the topological refactoring strategy (D) the topology of the native G-CSF was rewired from around the fixed binding epitope, and then was further mutated to idealise the core residues (blue volume (E)) and residues distal from the binding epitope (orange crust (E)). Both strategies aimed at simplifying the topology, reducing the size, and rigidifying the bound epitope conformation through alternate means. Figure 1 Disclosures No relevant conflicts of interest to declare.

The structural basis for signal promiscuity in a bacterial chemoreceptor.

Signalling through chemosensory pathways is typically initiated by the binding of signal molecules to the chemoreceptor ligand binding domain (LBD). The PcaY_PP chemoreceptor from Pseudomonasputida KT2440 is characterized by an unusually broad signal range, and minimal requisites for signal binding are the presence of a C6-membered ring and that of a carboxyl group. Previous studies have shown that only some of the multiple signals recognized by this chemoreceptor are of apparent metabolic value. We report here high-resolution structures of PcaY_PP-LBD in the absence and presence of four cognate chemoeffectors and glycerol. The domain formed a four-helix bundle (4HB), and both ligand binding sites of the dimer were occupied with the high-affinity ligands protocatechuate and quinate, whereas the lower-affinity ligands benzoate and salicylate were present in only one site. Ligand binding was verified by microcalorimetric titration of site-directed mutants revealing important roles of an arginine and number of polar residues that establish an extensive hydrogen bonding network with bound ligands. The comparison of the apo and holo structures did not provide evidence for this receptor employing a transmembrane signalling mechanism that involves piston-like shifts of the final helix. Instead, ligand binding caused rigid-body scissoring movements of both monomers of the dimer. Comparisons with the 4HB domains of the Tar and Tsr chemoreceptors revealed significant structural differences. Importantly, the ligand binding site in PcaY_PP-LBD is approximately 8Å removed from that of the Tar and Tsr receptors. Data indicate a significant amount of structural and functional diversity among 4HB domains. DATABASES: The coordinates and structure factors have been deposited in the protein data band with the following IDs: 6S1A (apo form), 6S18 (bound glycerol), 6S33 (bound protocatechuate), 6S38 (bound quinate), 6S3B (bound benzoate) and 6S37 (bound salicylate).

Structure of Epstein-Barr virus tegument protein complex BBRF2-BSRF1 reveals its potential role in viral envelopment

Epstein-Barr virus (EBV) is a γ-herpesvirus associated with the occurrence of several human malignancies. BBRF2 and BSRF1 are two EBV tegument proteins that have been suggested to form a hetero-complex and mediate viral envelopment, but the molecular basis of their interaction and the functional mechanism of this complex remains unknown. Here, we present crystal structures of BBRF2 alone and in complex with BSRF1. BBRF2 has a compact globular architecture featuring a central β-sheet that is surrounded by 10 helices, it represents a novel fold distinct from other known protein structures. The central portion of BSRF1 folds into two tightly associated antiparallel α-helices, forming a composite four-helix bundle with two α-helices from BBRF2 via a massive hydrophobic network. In vitro, a BSRF1-derived peptide binds to BBRF2 and reduces the number of viral genome copies in EBV-positive cells. Exogenous BBRF2 and BSRF1 co-localize at the Golgi apparatus. Furthermore, BBRF2 binds capsid and capsid-associated proteins, whereas BSRF1 associates with glycoproteins. These findings indicate that the BBRF2-BSRF1 complex tethers EBV nucleocapsids to the glycoprotein-enriched Golgi membrane, facilitating secondary envelopment.

The RING domain of TRIM69 promotes higher-order assembly.

Members of the TRIM protein family have been shown to inhibit a range of viral infections. Recently, TRIM69 was identified as a potent inhibitor of Vesicular stomatitis Indiana virus infection, with its inhibition being dependent upon multimerization. Using SEC-MALLS analysis, it is demonstrated that the assembly of TRIM69 is mediated through the RING domain and not the Bbox domain as has been shown for other TRIM proteins. Using X-ray crystallography, the structure of the TRIM69 RING domain has been determined to a resolution of 2.1 Å, the oligomerization interface has been identified and regions outside the four-helix bundle have been observed to form interactions that are likely to support assembly.

Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.