Evidence for Non-Essential Salt Bridges in the M-Gates of Mitochondrial Carrier Proteins.

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.

Memdock: An α-Helical Membrane Protein Docking Algorithm.

Memdock is a tool for docking α-helical membrane proteins which takes into consideration the lipid bilayer environment. Given two α-helical membrane located protein molecules, the method outputs a list of potential complexes sorted by energy criteria. The program includes three steps: docking, refinement, and re-ranking of the results. All three docking steps have been customized to the membrane environment in order to improve performance and reduce program run-time. In this chapter, we describe the application of our web server, referred to as Memdock, for prediction of the docking complex for a pair of input membrane protein structures. Memdock is freely available for academic users without registration at http://bioinfo3d.cs.tau.ac.il/Memdock/index.html .

X-ray Crystallographic Structure of α-Helical Peptide Stabilized by Hydrocarbon Stapling at i,i + 1 Positions.

Hydrocarbon stapling is a useful tool for stabilizing the secondary structure of peptides. Among several methods, hydrocarbon stapling at i,i + 1 positions was not extensively studied, and their secondary structures are not clarified. In this study, we investigate i,i + 1 hydrocarbon stapling between cis-4-allyloxy-l-proline and various olefin-tethered amino acids. Depending on the ring size of the stapled side chains and structure of the olefin-tethered amino acids, E- or Z-selectivities were observed during the ring-closing metathesis reaction (E/Z was up to 8.5:1 for 17-14-membered rings and up to 1:20 for 13-membered rings). We performed X-ray crystallographic analysis of hydrocarbon stapled peptide at i,i + 1 positions. The X-ray crystallographic structure suggested that the i,i + 1 staple stabilizes the peptide secondary structure to the right-handed α-helix. These findings are especially important for short oligopeptides because the employed stapling method uses two minimal amino acid residues adjacent to each other.

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles.

Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell's metabolism for the benefit of the bacterium to bypassing the host's defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.

A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures.

Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence-structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that determine the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong positive cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1.

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-ß or mixed-α/ß protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.

Concentration-Dependent Structural Transition of the HIV-1 gp41 MPER Peptide into α-Helical Trimers.

The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660-674 ) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2 O than H2 O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13 Cα chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.

Structural Basis for α-Helix Mimicry and Inhibition of Protein-Protein Interactions with Oligourea Foldamers.

Efficient optimization of a peptide lead into a drug candidate frequently needs further transformation to augment properties such as bioavailability. Among the different options, foldamers, which are sequence-based oligomers with precise folded conformation, have emerged as a promising technology. We introduce oligourea foldamers to reduce the peptide character of inhibitors of protein-protein interactions (PPI). However, the precise design of such mimics is currently limited by the lack of structural information on how these foldamers adapt to protein surfaces. We report a collection of X-ray structures of peptide-oligourea hybrids in complex with ubiquitin ligase MDM2 and vitamin D receptor and show how such hybrid oligomers can be designed to bind with high affinity to protein targets. This work should enable the generation of more effective foldamer-based disruptors of PPIs in the context of peptide lead optimization.

A comparison between internal protein nanoenvironments of α-helices and ß-sheets.

Secondary structure elements are generally found in almost all protein structures revealed so far. In general, there are more ß-sheets than α helices found inside the protein structures. For example, considering the PDB, DSSP and Stride definitions for secondary structure elements and by using the consensus among those, we found 60,727 helices in 4,376 chains identified in all-α structures and 129,440 helices in 7,898 chains identified in all-α and α + ß structures. For ß-sheets, we identified 837,345 strands in 184,925 ß-sheets located within 50,803 chains of all-ß structures and 1,541,961 strands in 355,431 ß-sheets located within 86,939 chains in all-ß and α + ß structures (data extracted on February 1, 2019). In this paper we would first like to address a full characterization of the nanoenvironment found at beta sheet locations and then compare those characteristics with the ones we already published for alpha helical secondary structure elements. For such characterization, we use here, as in our previous work about alpha helical nanoenvironments, set of STING protein structure descriptors. As in the previous work, we assume that we will be able to prove that there is a set of protein structure parameters/attributes/descriptors, which could fully describe the nanoenvironment around beta sheets and that appropriate statistically analysis will point out to significant changes in values for those parameters when compared for loci considered inside and outside defined secondary structure element. Clearly, while the univariate analysis is straightforward and intuitively understood, it is severely limited in coverage: it could be successfully applied at best in up to 25% of studied cases. The indication of the main descriptors for the specific secondary structure element (SSE) by means of the multivariate MANOVA test is the strong statistical tool for complete discrimination among the SSEs, and it revealed itself as the one with the highest coverage. The complete description of the nanoenvironment, by analogy, might be understood in terms of describing a key lock system, where all lock mini cylinders need to combine their elevation (controlled by a matching key) to open the lock. The main idea is as follows: a set of descriptors (cylinders in the key-lock example) must precisely combine their values (elevation) to form and maintain a specific secondary structure element nanoenvironment (a required condition for a key being able to open a lock).

Exposing the distinctive modular behavior of ß-strands and α-helices in folded proteins.

Although folded proteins are commonly depicted as simplistic combinations of ß-strands and α-helices, the actual properties and functions of these secondary-structure elements in their native contexts are just partly understood. The principal reason is that the behavior of individual ß- and α-elements is obscured by the global folding cooperativity. In this study, we have circumvented this problem by designing frustrated variants of the mixed α/ß-protein S6, which allow the structural behavior of individual ß-strands and α-helices to be targeted selectively by stopped-flow kinetics, X-ray crystallography, and solution-state NMR. Essentially, our approach is based on provoking intramolecular "domain swap." The results show that the α- and ß-elements have quite different characteristics: The swaps of ß-strands proceed via global unfolding, whereas the α-helices are free to swap locally in the native basin. Moreover, the α-helices tend to hybridize and to promote protein association by gliding over to neighboring molecules. This difference in structural behavior follows directly from hydrogen-bonding restrictions and suggests that the protein secondary structure defines not only tertiary geometry, but also maintains control in function and structural evolution. Finally, our alternative approach to protein folding and native-state dynamics presents a generally applicable strategy for in silico design of protein models that are computationally testable in the microsecond-millisecond regime.

Structural basis for inhibitory effects of Smad7 on TGF-ß family signaling.

Smad6 and Smad7 are classified as inhibitory Smads (I-Smads). They are crucial in the fine-tuning of signals by cytokines of the transforming growth factor-ß (TGF-ß) family. They are negative feedback regulators and principally target the activated type I receptors as well as the activated Smad complexes, but with distinct specificities. Smad7 inhibits Smad signaling from all seven type I receptors of the TGF-ß family, whereas Smad6 preferentially inhibits Smad signaling from the bone morphogenetic protein (BMP) type I receptors, BMPR1A and BMPR1B. The target specificities are attributed to the C-terminal MH2 domain. Notably, Smad7 utilizes two alternative molecular surfaces for its inhibitory function against type I receptors. One is a basic groove composed of the first α-helix and the L3 loop, a structure that is shared with Smad6 and receptor-regulated Smads (R-Smads). The other is a three-finger-like structure (consisting of residues 331-361, 379-387, and the L3 loop) that is unique to Smad7. The underlying structural basis remains to be elucidated in detail. Here, we report the crystal structure of the MH2 domain of mouse Smad7 at 1.9 Å resolution. The three-finger-like structure is stabilized by a network of hydrogen bonds between residues 331-361 and 379-387, thus forming a molecular surface unique to Smad7. Furthermore, we discuss how Smad7 antagonizes the activated Smad complexes composed of R-Smad and Smad4, a common partner Smad.

Structural and functional characterization of the pore-forming domain of pinholin S2168.

Pinholin S2168 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual "pinholes." Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state 15N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (Smol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G40xxxS44xxxG48) together with a patch of H-bonding residues (T51, T54, N55) sideways along TMD2, available for helix-helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Functional Characterization of Temporin-SHe, a New Broad-Spectrum Antibacterial and Leishmanicidal Temporin-SH Paralog from the Sahara Frog (Pelophylax saharicus).

Amphibian skin is a promising natural resource for antimicrobial peptides (AMPs), key effectors of innate immunity with attractive therapeutic potential to fight antibiotic-resistant pathogens. Our previous studies showed that the skin of the Sahara Frog (Pelophylax saharicus) contains broad-spectrum AMPs of the temporin family, named temporins-SH. Here, we focused our study on temporin-SHe, a temporin-SHd paralog that we have previously identified in this frog but was never structurally and functionally characterized. We synthesized and determined the structure of temporin-SHe. This non-amphipathic α-helical peptide was demonstrated to strongly destabilize the lipid chain packing of anionic multilamellar vesicles mimicking bacterial membranes. Investigation of the antimicrobial activity revealed that temporin-SHe targets Gram-negative and Gram-positive bacteria, including clinical isolates of multi-resistant Staphylococcus aureus strains. Temporin-SHe exhibited also antiparasitic activity toward different Leishmania species responsible for visceral leishmaniasis, as well as cutaneous and mucocutaneous forms. Functional assays revealed that temporin-SHe exerts bactericidal effects with membrane depolarization and permeabilization, via a membranolytic mechanism observed by scanning electron microscopy. Temporin-SHe represents a new member of the very limited group of antiparasitic temporins/AMPs. Despite its cytotoxicity, it is nevertheless an interesting tool to study the AMP antiparasitic mechanism and design new antibacterial/antiparasitic agents.

Cytotoxicity of α-Helical, Staphylococcus aureus PSMα3 Investigated by Post-Ion-Mobility Dissociation Mass Spectrometry.

Our knowledge of amyloid formation and cytotoxicity originating from self-assembly of α-helical peptides is incomplete. PSMα3 is the only system where high-resolution X-ray crystallography and toxicity data are available. Oligomers of multiple α-helical monomers are less stable than those of ß-strands, partially due to the lack of a consistent hydrogen-bonding network. It is challenging to preserve such oligomers in the gas phase where mass-selected structural studies using ion-mobility spectrometry mass spectrometry (IMS-MS) could be performed. As the oligomers fall apart after exiting the drift cell of the mass spectrometer, novel features that have shorter (a loss of charged species) or longer (a loss of neutral species) arrival times than expected are present together with those from the intact species. By obtaining a complete data set of PSMα3 peptides in solution and with n-dodecyl-ß-d-maltoside, a micelle-forming detergent, we are able to discern the dissociated from the intact oligomers and detergent-bound complexes and correlate the reported cytotoxicity to the peptide oligomeric structures and their interactions with membrane mimetics. The study sheds new insights into the interpretation of IMS-MS data from biomolecular self-assembly studies-an important and timely topic.

The variable oligomeric state of Amuc_1100 from Akkermansia muciniphila.

Akkermansia muciniphila is a beneficial microorganism colonized in the human gut that can reverse many intestinal metabolic-related diseases. Amuc_1100 is an outer-membrane protein of A. muciniphila. Oral administration of Amuc_1100 can reduce fat mass development, insulin resistance, and dyslipidemia in mice and activated the toll-like receptor 2 (TLR2) to regulate the immune response of the host, but the molecular mechanism remains unclear. Here we report the crystal structure of the extramembranous domain of Amuc_1100, which consists of a four-stranded antiparallel ß-sheet and four α-helices. Two C-terminal helices and the four-stranded antiparallel ß-sheet formed two "αßß" motifs and constituted the core domain, which shared a similar fold with type IV pili and type II Secretion system protein. Although the full-length of the extramembranous domain of Amuc_1100 existed as a monomer in solution, they formed trimer in the crystal. Elimination of the N-terminal coiled-coil helix α1 led to dimerization of Amuc_1100 both in solution and in crystal, indicating that the oligomeric state of Amuc_1100 was variable and could be influenced by α1. In addition, we identified that Amuc_1100 could directly bind human TLR2 (hTRL2) in vitro, suggesting that Amuc_1100 may serve as a new ligand for hTLR2. Dimerization of Amuc_1100 improved its hTLR2-binding affinity, suggesting that the α1-truncated Amuc_1100 could be a beneficial candidate for the development of A. muciniphila related drugs.

Helix stabilization by stapled N-capping box.

The N-capping box is a distinct helix-stabilizing motif frequently found in proteins. In this study, we examined a ruthenium-mediated intramolecular backbone to side chain macrocyclization as a rigidified mimicry of the N-capping box. Experimental data indicate that the 15-membered macrocycle formed by a hept-4-enoyl staple, which directly tethers the α-amino group of N1 residue and the α-carbon of N3 residue, is highly effective in stabilizing helical structures of short peptides.

Robust De Novo-Designed Homotetrameric Coiled Coils.

De novo-designed protein domains are increasingly being applied in biotechnology, cell biology, and synthetic biology. Therefore, it is imperative that these proteins be robust to superficial changes; i.e., small changes to their amino acid sequences should not cause gross structural changes. In turn, this allows properties such as stability and solubility to be tuned without affecting structural attributes like tertiary fold and quaternary interactions. Reliably designed proteins with predictable behaviors may then be used as scaffolds to incorporate function, e.g., through the introduction of features for small-molecule, metal, or macromolecular binding, and enzyme-like active sites. Generally, achieving this requires the starting protein fold to be well understood. Herein, we focus on designing α-helical coiled coils, which are well studied, widespread, and often direct protein-protein interactions in natural systems. Our initial investigations reveal that a previously designed parallel, homotetrameric coiled coil, CC-Tet, is not robust to sequence changes that were anticipated to maintain its structure. Instead, the alterations switch the oligomeric state from tetramer to trimer. To improve the robustness of designed homotetramers, additional sequences based on CC-Tet were produced and characterized in solution and by X-ray crystallography. Of these updated sequences, one is robust to truncation and to changes in surface electrostatics; we call this CC-Tet*. Variants of the general CC-Tet* design provide a set of homotetrameric coiled coils with unfolding temperatures in the range from 40 to >95 °C. We anticipate that these will be of use in applications requiring robust and well-defined tetramerization domains.

Helix 8 is the essential structural motif of mechanosensitive GPCRs.

G-protein coupled receptors (GPCRs) are versatile cellular sensors for chemical stimuli, but also serve as mechanosensors involved in various (patho)physiological settings like vascular regulation, cardiac hypertrophy and preeclampsia. However, the molecular mechanisms underlying mechanically induced GPCR activation have remained elusive. Here we show that mechanosensitive histamine H1 receptors (H1Rs) are endothelial sensors of fluid shear stress and contribute to flow-induced vasodilation. At the molecular level, we observe that H1Rs undergo stimulus-specific patterns of conformational changes suggesting that mechanical forces and agonists induce distinct active receptor conformations. GPCRs lacking C-terminal helix 8 (H8) are not mechanosensitive, and transfer of H8 to non-responsive GPCRs confers, while removal of H8 precludes, mechanosensitivity. Moreover, disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Altogether, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in (patho)physiology.

SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure.

SLC6A14 (ATB0,+) is a sodium- and chloride-dependent neutral and dibasic amino acid transporter that regulates the distribution of amino acids across cell membranes. The transporter is overexpressed in many human cancers characterized by an increased demand for amino acids; as such, it was recently acknowledged as a novel target for cancer therapy. The knowledge on the molecular mechanism of SLC6A14 transport is still limited, but some elegant studies on related transporters report the involvement of the 12 transmembrane α-helices in the transport mechanism, and describe structural rearrangements mediated by electrostatic interactions with some pivotal gating residues. In the present work, we constructed a SLC6A14 model in outward-facing conformation via homology modeling and used molecular dynamics simulations to predict amino acid residues critical for substrate recognition and translocation. We docked the proteinogenic amino acids and other known substrates in the SLC6A14 binding site to study both gating regions and the exposed residues involved in transport. Interestingly, some of these residues correspond to those previously identified in other LeuT-fold transporters; however, we could also identify a novel relevant residue with such function. For the first time, by combined approaches of molecular docking and molecular dynamics simulations, we highlight the potential role of these residues in neutral amino acid transport. This novel information unravels new aspects of the human SLC6A14 structure-function relationship and may have important outcomes for cancer treatment through the design of novel inhibitors of SLC6A14-mediated transport.