Fast native-SAD phasing for routine macromolecular structure determination.

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation.

Ca(2+) efflux by Ca(2+) cation antiporter (CaCA) proteins is important for maintenance of Ca(2+) homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na(+)/Ca(2+) exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca(2+) efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca(2+)/H(+) antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca(2+)-binding site. A hydrophobic "seal" closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca(2+)/H(+) exchange pathway. In the Ca(2+)-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na(+)-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca(2+)/H(+) exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.

PRIGo: a new multi-axis goniometer for macromolecular crystallography.

The Parallel Robotics Inspired Goniometer (PRIGo) is a novel compact and high-precision goniometer providing an alternative to (mini-)kappa, traditional three-circle goniometers and Eulerian cradles used for sample reorientation in macromolecular crystallography. Based on a combination of serial and parallel kinematics, PRIGo emulates an arc. It is mounted on an air-bearing stage for rotation around ω and consists of four linear positioners working synchronously to achieve x, y, z translations and χ rotation (0-90°), followed by a ϕ stage (0-360°) for rotation around the sample holder axis. Owing to the use of piezo linear positioners and active correction, PRIGo features spheres of confusion of <1 µm, <7 µm and <10 µm for ω, χ and ϕ, respectively, and is therefore very well suited for micro-crystallography. PRIGo enables optimal strategies for both native and experimental phasing crystallographic data collection. Herein, PRIGo hardware and software, its calibration, as well as applications in macromolecular crystallography are described.

Structural insights into VirB-DNA complexes reveal mechanism of transcriptional activation of virulence genes.

VirB activates transcription of virulence genes in Shigella flexneri by alleviating heat-stable nucleoid-structuring protein-mediated promoter repression. VirB is unrelated to the conventional transcriptional regulators, but homologous to the plasmid partitioning proteins. We determined the crystal structures of VirB HTH domain bound by the cis-acting site containing the inverted repeat, revealing that the VirB-DNA complex is related to ParB-ParS-like complexes, presenting an example that a ParB-like protein acts exclusively in transcriptional regulation. The HTH domain of VirB docks DNA major groove and provides multiple contacts to backbone and bases, in which the only specific base readout is mediated by R167. VirB only recognizes one half site of the inverted repeats containing the most matches to the consensus for VirB binding. The binding of VirB induces DNA conformational changes and introduces a bend at an invariant A-tract segment in the cis-acting site, suggesting a role of DNA remodeling. VirB exhibits positive cooperativity in DNA binding that is contributed by the C-terminal domain facilitating VirB oligomerization. The isolated HTH domain only confers partial DNA specificity. Additional determinants for sequence specificity may reside in N- or C-terminal domains. Collectively, our findings support and extend a previously proposed model for relieving heat-stable nucleoid-structuring protein-mediated repression by VirB.

Short-peptide fusion inhibitors with high potency against wild-type and enfuvirtide-resistant HIV-1.

Peptides derived from the C-terminal heptad repeat (C peptides) of HIV-1 gp41 are potent inhibitors against virus entry. However, development of a short C peptide possessing high anti-HIV potency is considered a daunting challenge. We recently discovered that the residues Met626 and Thr627 preceding the pocket-binding domain of the C peptide adopt a unique M-T hook structure that is crucial for the design of HIV-1 fusion inhibitors. In this study, we first presented a proof-of-concept prototype that the M-T hook residues can dramatically improve the antiviral activity and thermostability of a short C peptide. We then generated a 24-mer peptide termed MT-SC22EK by incorporating the M-T hook structure to the N terminus of the poorly active short C peptide SC22EK. Amazingly, MT-SC22EK inhibited HIV-1-mediated cell fusion and infection at a level comparable to C34, T1249, SC29EK, and sifuvirtide, and it was highly active against diverse HIV-1 subtypes and variants, including those T20 (enfuvirtide) and SC29EK-resistant viruses. The high-resolution crystal structure of MT-SC22EK reveals the N-terminal M-T hook conformation folded by incorporated Met626 and Thr627 and identifies the C-terminal boundary critical for the anti-HIV activity. Collectively, our studies provide new insights into the mechanisms of HIV-1 fusion and its inhibition.

Broad antiviral activity and crystal structure of HIV-1 fusion inhibitor sifuvirtide.

Sifuvirtide (SFT) is an electrostatically constrained α-helical peptide fusion inhibitor showing potent anti-HIV activity, good safety, and pharmacokinetic profiles, and it is currently under phase II clinical trials in China. In this study, we demonstrate its potent and broad anti-HIV activity by using diverse HIV-1 subtypes and variants, including subtypes A, B, and C that dominate the AIDS epidemic worldwide, and subtypes B', CRF07_BC, and CRF01_AE recombinants that are currently circulating in China, and those possessing cross-resistance to the first and second generation fusion inhibitors. To elucidate its mechanism of action, we determined the crystal structure of SFT in complex with its target N-terminal heptad repeat region (NHR) peptide (N36), which fully supports our rational inhibitor design and reveals its key motifs and residues responsible for the stability and anti-HIV activity. As anticipated, SFT adopts fully helical conformation stabilized by the multiple engineered salt bridges. The designing of SFT also provide novel inter-helical salt bridges and hydrogen bonds that improve the affinity of SFT to NHR trimer. The extra serine residue and acetyl group stabilize α-helicity of the N-terminal portion of SFT, whereas Thr-119 serves to stabilize the hydrophobic NHR pocket. In addition, our structure demonstrates that the residues critical for drug resistance, located at positions 37, 38, 41, and 43 of NHR, are irreplaceable for maintaining the stable fusogenic six-helix bundle structure. Our data present important information for developing SFT for clinical use and for designing novel HIV fusion inhibitors.

The M-T hook structure is critical for design of HIV-1 fusion inhibitors.

CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors.

Structural basis of potent and broad HIV-1 fusion inhibitor CP32M.

CP32M is a newly designed peptide fusion inhibitor possessing potent anti-HIV activity, especially against T20-resistant HIV-1 strains. In this study, we show that CP32M can efficiently inhibit a large panel of diverse HIV-1 variants, including subtype B', CRF07_BC, and CRF01_AE recombinants and naturally occurring or induced T20-resistant viruses. To elucidate its mechanism of action, we determined the crystal structure of CP32M complexed with its target sequence. Differing from its parental peptide, CP621-652, the (621)VEWNEMT(627) motif of CP32M folds into two α-helix turns at the N terminus of the pocket-binding domain, forming a novel layer in the six-helix bundle structure. Prominently, the residue Asn-624 of the (621)VEWNEMT(627) motif is engaged in the polar interaction with a hydrophilic ridge that borders the hydrophobic pocket on the N-terminal coiled coil. The original inhibitor design of CP32M provides several intra- and salt bridge/hydrogen bond interactions favoring the stability of the helical conformation of CP32M and its interactions with N-terminal heptad repeat (NHR) targets. We identified a novel salt bridge between Arg-557 on the NHR and Glu-648 of CP32M that is critical for the binding of CP32M and resistance against the inhibitor. Therefore, our data present important information for developing novel HIV-1 fusion inhibitors for clinical use.

Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652.

The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the (621)QIWNNMT(627) motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621-652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621-652 complexed by T21. We find that the (621)QIWNNMT(627) motif does not maintain the α-helical conformation. Instead, residues Met(626) and Thr(627) form a unique hook-like structure (denoted as M-T hook), in which Thr(627) redirects the peptide chain to position Met(626) above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met(626) caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621-652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition.

Structural characterization of the ribonuclease H-like type ASKHA superfamily kinase MK0840 from Methanopyrus kandleri.

Murein recycling is a process in which microorganisms recover peptidoglycan-degradation products in order to utilize them in cell wall biosynthesis or basic metabolic pathways. Methanogens such as Methanopyrus kandleri contain pseudomurein, which differs from bacterial murein in its composition and branching. Here, four crystal structures of the putative sugar kinase MK0840 from M. kandleri in apo and nucleotide-bound states are reported. MK0840 shows high similarity to bacterial anhydro-N-acetylmuramic acid kinase, which is involved in murein recycling. The structure shares a common fold with panthothenate kinase and the 2-hydroxyglutaryl-CoA dehydratase component A, both of which are members of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases. Local conformational changes in the nucleotide-binding site between the apo and holo forms are observed upon nucleotide binding. Further insight is given into domain movements and putative active-site residues are identified.

Phosphoenolpyruvate: sugar phosphotransferase system from the hyperthermophilic Thermoanaerobacter tengcongensis.

Thermoanaerobacter tengcongensis is a thermophilic eubacterium that has a phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) of 22 proteins. The general PTS proteins, enzyme I and HPr, and the transporters for N-acetylglucosamine (EIICB(GlcNAc)) and fructose (EIIBC(Fru)) have thermal unfolding transitions at ∼90 °C and a temperature optimum for in vitro sugar phosphotransferase activity of 65 °C. The phosphocysteine of a EIICB(GlcNAc) mutant is unusually stable at room temperature with a t(1/2) of 60 h. The PEP binding C-terminal domain of enzyme I (EIC) forms a metastable covalent adduct with PEP at 65 °C. Crystallization of this adduct afforded the 1.68 Å resolution structure of EIC with a molecule of pyruvate in the active site. We also report the 1.83 Å crystal structure of the EIC-PEP complex. The comparison of the two structures with the apo form and with full-length EI shows differences between the active site side chain conformations of the PEP and pyruvate states but not between the pyruvate and apo states. In the presence of PEP, Arg465 forms a salt bridge with the phosphate moiety while Glu504 forms salt bridges with Arg186 and Arg195 of the N-terminal domain of enzyme I (EIN), which stabilizes a conformation appropriate for the in-line transfer of the phosphoryl moiety from PEP to His191. After transfer, Arg465 swings 4.8 Å away to form an alternative salt bridge with the carboxylate of Glu504. Glu504 loses the grip of Arg186 and Arg195, and the EIN domain can swing away to hand on the phosphoryl group to the phosphoryl carrier protein HPr.

Structure and function of CinD (YtjD) of Lactococcus lactis, a copper-induced nitroreductase involved in defense against oxidative stress.

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k(cat) values of 27 and 11 s(-1), respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 A and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.

Classical swine fever virus can remain virulent after specific elimination of the interferon regulatory factor 3-degrading function of Npro.

Pestiviruses prevent alpha/beta interferon (IFN-alpha/beta) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral N(pro) nonstructural protein. N(pro) is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire N(pro) gene resulted in attenuation in pigs. In order to elaborate on the role of the N(pro)-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in N(pro) that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of N(pro). We describe two mutations in N(pro) that eliminate N(pro)-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for N(pro) to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-alpha/beta induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-alpha/beta induction is not a prerequisite for CSFV virulence.

Crystal structures of archaemetzincin reveal a moldable substrate-binding site.

BACKGROUND: Archaemetzincins are metalloproteases occurring in archaea and some mammalia. They are distinct from all the other metzincins by their extended active site consensus sequence HEXXHXXGXXHCX(4)CXMX(17)CXXC featuring four conserved cysteine residues. Very little is known about their biological importance and structure-function relationships. PRINCIPAL FINDINGS: Here we present three crystal structures of the archaemetzincin AfAmzA (Uniprot O29917) from Archaeoglobus fulgidus, revealing a metzincin architecture featuring a zinc finger-like structural element involving the conserved cysteines of the consensus motif. The active sites in all three structures are occluded to different extents rendering the enzymes proteolytically inactive against a large variety of tested substrates. Owing to the different ligand binding there are significant differences in active site architecture, revealing a large flexibility of the loops covering the active site cleft. CONCLUSIONS: The crystal structures of AfAmzA provide the structural basis for the lack of activity in standard proteolytic assays and imply a triggered activity onset upon opening of the active site cleft.