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.

Pestiviruses prevent alpha/beta interferon (IFN-␣/␤) 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-␣/␤ 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-␣/␤ induction is not a prerequisite for CSFV virulence.

Background:We recently found that N-terminal residues Met-626 and Thr-627 of HIV-1 fusion inhibitor CP621-652 adopt a unique hook-like structure, termed the M-T hook. Results: The structure and function of the M-T hook have been characterized. Conclusion: The M-T hook is critical for the stability and antiviral activity of HIV-1 fusion inhibitors. Significance: Our data provide important information for designing novel HIV-1 fusion inhibitors.

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.

CaCAs are integral membrane proteins containing 10-11 predicted transmembrane helices (TMs) (1) (SI Appendix, Fig. S1). Despite the divergence in cation driving force, the superfamily is defined by the presence of two highly conserved α-repeat motifs in TMs 2-3 (α-1) and TMs 7-8 (α-2). The importance of these conserved motifs for Ca 2+ /cation exchange has been well documented in all characterized CaCA proteins (8-10), implying the conservation of the Ca 2+ /cation translocation mechanism in the CaCA superfamily. Recently, Liao et al. reported the crystal structure of the NCX_Mj protein (6). The structure shows a large cavity opening on the periplasmic surface (outward-facing conformation) with both Ca 2+ and Na + bound at the α-repeat regions, suggesting the following hypotheses: (i) a substrate alternating access mechanism in which Ca 2+ and the counter transported cation access the ion-binding sites from each side of the membrane surface; and (ii) transition between outward-facing and inward-facing conformations is required to achieve Ca 2+ /cation exchange. Although the structure of the outward-facing conformation of NCX_Mj is available, structural evidence for the conformational change essential for alternating substrate access has not yet been obtained for any CaCA member. In addition, how the CaCA superfamily proteins modify their Ca 2+ -translocation pathways to use the different cation-gradient driving forces, H + vs. Na + , remains a fundamental and interesting question.To address these important questions, here, we report the crystal structure of an inward-facing conformation of the YfkE protein. This structure, together with protein kinetic analysis, not only provides an important structural characterization of the mechanism of Ca 2+ efflux across the cell membrane but also sheds light on the different modes of energy coupling used by members of the CaCA protein superfamily. Results Ca 2+-Transport Specificity and Structural Determination of YfkE. YfkE catalyzes a H+ -coupled Ca 2+ influx in everted vesicles (SI Appendix, Fig. S2A This article is a PNAS Direct Submission.Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4KJR and 4KJS).