Solution structure of the cytoplasmic domain of NhaP2 a K+/H+ antiporter from Vibrio cholera.

NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.

Solution structure and oligomeric state of the E. coliglycerol facilitator.

Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.

Impact of double-stranded RNA characteristics on the activation of human 2'-5'-oligoadenylate synthetase 2 (OAS2).

Human 2'-5' oligoadenylate synthetases (OAS) are a family of interferon-inducible proteins that, upon activation by double-stranded RNA, polymerize ATP into 2'-5' linked oligoadenylates. In this study, we probed the RNA cofactor specificity of the two smallest isozymes, OAS1 and OAS2. First, we developed a strategy for the expression and purification of recombinant human OAS2 from eukaryotic cells and quantified the activity of the enzyme relative to OAS1 in vitro. We then confirmed that both OAS2 domains, as opposed to only the domain containing the canonical catalytic aspartic acid triad, are required for enzymatic activity. Enzyme kinetics of both OAS1 and OAS2 in the presence of a variety of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While in this study OAS1 can be catalytically activated by dsRNA of any length greater than 19 bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35 bp. Interestingly, activation of OAS2 was also more efficient when the dsRNA contained 3'-overhangs, despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

Correction: Dramatic and concerted conformational changes enable rhodocetin to block α2ß1 integrin selectively.

[This corrects the article DOI: 10.1371/journal.pbio.2001492.].

Dramatic and concerted conformational changes enable rhodocetin to block α2ß1 integrin selectively.

The collagen binding integrin α2ß1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αß and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2ß1 integrin. Besides the release of the nonbinding RCαß tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the "closed" conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2ß1 integrin.

Structural Decoding of the Netrin-1/UNC5 Interaction and its Therapeutical Implications in Cancers.

Netrin-1 has been shown to be up-regulated in a fraction of human cancers as a mechanism to allow these tumors to escape the pro-apoptotic activity of some of its main dependence receptors, the UNC5 homologs (UNC5H). Here we identify the V-2 domain of netrin-1 to be important for its interaction with the Ig1/Ig2 domains of UNC5H2. We generate a humanized anti-netrin-1 antibody that disrupts the interaction between netrin-1 and UNC5H2 and triggers death of netrin-1-expressing tumor cells in vitro. We also present evidence that combining the anti-netrin-1 antibody with epidrugs such as decitabine could be effective in treating tumors showing no or modest netrin-1 expression. These results support that this antibody is a promising drug candidate.

The C-terminal cytoplasmic portion of the NhaP2 cation-proton antiporter from Vibrio cholerae affects its activity and substrate affinity.

In this work, we report the phenotypic and biochemical effects of deleting the C-terminal cytoplasmic portion of the NhaP2 cation/proton antiporter from Vibrio cholerae. While the deletion changed neither the expression nor targeting of the Vc-NhaP2 in an antiporter-less Escherichia coli strain, it resulted in a changed sensitivity of the host to sodium ions at neutral pH, indicating an altered Na(+) transport through the truncated variant. When assayed in inside-out sub-bacterial vesicles, the truncation was found to result in greatly reduced K(+)/H(+) and Na(+)/H(+) antiport activity at all pH values tested and a greater than fivefold decrease in the affinity for K(+) (measured as the apparent K m) at pH 7.5. Being expressed in trans in a strain of V. cholerae bearing a chromosomal nhaP2 deletion, the truncated nhaP2 gene was able to complement its inability to grow in potassium-rich medium at pH 6.0. Thus the residual K(+)/H(+) antiport activity associated with the truncated Vc-NhaP2 was still sufficient to protect cells from an over-accumulation of K(+) ions in the cytoplasm. The presented data suggest that while the cytoplasmic portion of Vc-NhaP2 is not involved in ion translocation directly, it is necessary for optimal activity and substrate binding of the Vc-NhaP2 antiporter.

The pentameric channel of COMPcc in complex with different fatty acids.

BACKGROUND: COMPcc forms a pentameric left-handed coiled coil that is known to bind hydrophilic signaling molecules such as vitamin D(3), and vitamin A. PRINCIPAL FINDINGS: In an integrated approach we reveal the unique binding properties of COMPcc for saturated and unsaturated fatty acids. Our observations suggest that residues Met33 (gating pore), Thr40/Asn41 (water chamber) and Gln54 (electrostatic trap) are key elements for the binding of fatty acids by COMPcc. In addition, this work characterizes the binding of various fatty acids to COMPcc using fluorescence spectroscopy. Our findings reveal a binding trend within the hydrophobic channel of COMPcc, namely, that is driven by length of the methylene tail and incorporation of unsaturation. CONCLUSION/SIGNIFICANCE: The unique binding properties imply that COMPcc may be involved in signalling functions in which hydrophilic ligands are involved. The pentameric channel is a unique carrier for lipophilic compounds. This opens the exciting possibility that COMPcc could be developed as a targeted drug delivery system.

Absence of a catalytic water confers resistance to the neurotoxin gabaculine.

Gabaculine is a potent inhibitor of the vitamin B6-dependent key enzyme in chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase (GSAM). The inhibition effect is caused by an enzymatic deprotonation of the neurotoxin and requires the aldimine (PLP) form of the cofactor at the active site. In this study, we show that a single-point mutation confers resistance to gabaculine. A combined functional and structural analysis of wild-type GSAM in complex with gabaculine and the GSAM(M248I) form allowed us to decipher in atomic detail the molecular basis of this unique resistance. Interestingly, the gabaculine tolerance is caused by the absence of an essential water molecule that has a dual functional role. It serves as a nucleophilic shuttle for the hydroxyl anion along the reaction pathway and holds active-site Lys273 in a catalytically competent conformation. The single-point mutant is not able to fix this catalytic water between the beta-branched side chain of Ile248 and Lys273. As a consequence, the mutant enzyme is trapped in a gabaculine-insensitive but still enzymatically active amine (PMP) form.

The use of coiled-coil proteins in drug delivery systems.

The coiled-coil motif is found in approximately 10% of all protein sequences and is responsible for the oligomerization of proteins in a highly specific manner. Coiled-coil proteins exhibit a large diversity of function (e.g. gene regulation, cell division, membrane fusion, drug extrusion) thus demonstrating the significance of oligomerization in biological systems. The classical coiled-coil domain comprises a series of consecutive heptad repeats in the protein sequence that are readily identifiable by the location of hydrophobic residues at the 'a' and 'd' positions. This gives rise to an alpha-helical structure in which between 2 and 7 helices are wound around each other in the form of a left-handed supercoil. More recently, structures of coiled-coil domains have been solved that have an 11 residue (undecad) or a 15 residue (pentadecad) repeat, which show the formation of a right-handed coiled-coil structure. The high stability of coiled coils, together with the presence of large internal cavities in the pentameric coiled-coil domain of cartilage oligomerization matrix protein (COMPcc) and the tetrameric right-handed coiled coil of Staphylothermus marinus (RHCC) has led us and others to look for therapeutic applications. In this review, we present evidence in support of a vitamin A and vitamin D(3) binding activity for the pentameric COMPcc molecule. In addition, we will discuss exciting new developments which show that the RHCC tetramer is capable of binding the major anticancer drug cisplatin and the ability to fuse it to an antigenic epitope for the development of a new generation of vaccines.

TatBC, TatB, and TatC form structurally autonomous units within the twin arginine protein transport system of Escherichia coli.

The Tat (twin arginine translocation) system transports folded proteins across bacterial and thylakoid membranes. The integral membrane proteins TatA, TatB, and TatC are the essential components of the Tat pathway in Escherichia coli. We demonstrate that formation of a stable complex between TatB and TatC does not require TatA or other Tat components. We show that the TatB and TatC proteins are each able to a form stable, defined, homomultimeric complexes. These we suggest correspond to structural subcomplexes within the parental TatBC complex. We infer that TatC forms a core to the TatBC complex on to which TatB assembles.

Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.

The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane alpha-helix that precedes an amphipathic alpha-helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.

Expression of functional recombinant von Willebrand factor-A domain from human complement C2: a potential binding site for C4 and CRIT.

CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the beta-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224-437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 alpha, beta and gamma chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4beta chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2-CRIT and C2-C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.

Crystal structure of the IIB(Sor) domain of the sorbose permease from Klebsiella pneumoniae solved to 1.75A resolution.

The phosphoenolpyruvate transferase system (PTS) is the major pathway by which bacteria import hexose sugars across the plasma membrane. The PTS transfers a phosphoryl group sequentially via several components from the glycolytic intermediate phosphoenolpyruvate (PEP) to the translocated sugar. It is comprised of the two general proteins enzyme I and HPr, and a sugar-specific enzyme II complex. Sugar translocation is through the membrane domain of the enzyme II complex. The enzyme II complex can belong to one of six families based upon sequence similarity, with the sorbose transporter from Klebsiella pneumoniae a member of the mannose family.The structure of the IIB(Sor) domain was solved to 1.75A resolution by molecular replacement. It has a central core of seven parallel beta-strands surrounded by a total of six alpha-helices. Three helices cover the front face, one the back face with the remaining two capping the central beta-sheet at the top and bottom. The catalytic His15 residue is situated on the surface-exposed loop between strand 1 and helix 1. In addition to the features previously observed in the homologous IIB(Lev) domain from Bacillus subtilis we see new features in the IIB(Sor) structure. First, the catalytic His15 side-chain is fixed in a specific conformation by forming a short hydrogen bond with Asp10, which in turn makes a salt-bridge with Arg8. Second, as observed in other phosphoproteins, an arginine residue (Arg12) is well poised to stabilize a phosphoryl group on His15. Third, we see an Asp/His pair reminiscent of that observed in the IIA(Man) domain from Escherichia coli. Finally, docking of IIA(Man) to IIB(Sor) shows that Arg12 in its current conformation is well positioned to assist the subsequent transfer of the phosphoryl group onto the sugar in line with previous mutagenesis studies.