Sandro Waltersperger

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.

Broad Antiviral Activity and Crystal Structure of HIV-1 Fusion Inhibitor Sifuvirtide

Background: Sifuvirtide (SFT) is an HIV peptide fusion inhibitor under phase II clinical trials. Results: Crystal structure of SFT complexed with gp41 NHR peptide and the potent activity of SFT against diverse HIV-1 variants were determined. Conclusion: Crystal structure fully supports earlier peptide design. Significance: Our data present important information for developing SFT for clinical use and for designing novel HIV fusion inhibitors. 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.

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.—Chong, H., Yao, X., Qiu, Z., Sun, J., Zhang, M., Waltersperger, S., Wang, M., Liu, S.‐L., Cui, S., and He, Y. Short‐peptide fusion inhibitors with high potency against wild‐type and enfuvirtide‐resistant HIV‐1. FASEB J. 27, 1203–1213 (2013). www.fasebj.org

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors

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. 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.

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

Background: CP621–652 is a HIV-1 fusion inhibitor peptide containing the gp41 621QIWNNMT627 motif. Results: The crystal structure of CP621–652 in complex with T21 was determined and mutational analyses were performed. Conclusion: The residues Met626 and Thr627 in the gp41 621QIWNNMT627 motif are critical for HIV-1 entry and inhibition. Significance: Our data provide important information for designing novel HIV fusion inhibitors. 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 621QIWNNMT627 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 621QIWNNMT627 motif does not maintain the α-helical conformation. Instead, residues Met626 and Thr627 form a unique hook-like structure (denoted as M-T hook), in which Thr627 redirects the peptide chain to position Met626 above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met626 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.

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

Ca2+ efflux by Ca2+ cation antiporter (CaCA) proteins is important for maintenance of Ca2+ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na+/Ca2+ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca2+ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca2+/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 Ca2+-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca2+/H+ exchange pathway. In the Ca2+-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 Ca2+/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 design and performance of the new multi-axis goniometer PRIGo developed at the Swiss Light Source at Paul Scherrer Institute is described.

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

Background: CP32M is a newly designed HIV-1 fusion inhibitor. Results: The crystal structure of CP32M and its potent activity against diverse HIV-1 variants were determined. Conclusion: The crystal structure reveals the mechanistic insights of CP32M. Significance: Our data provide important information for designing potent HIV-1 fusion inhibitors. 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 621VEWNEMT627 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 621VEWNEMT627 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.

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.

Native SAD Structure Solution from Merohedrally Twinned Data

Up until now, comparatively few structures were solved by native SAD. Recent advances in multi crystal averaging [1] have shown that native SAD can be applied to an increasing number of cases. Though theoretically possible [2], successful structure solutions from twinned data have not been reported yet. Here, we report the structure solution of the human Centromere protein M from a merohedrally twinned crystal with a twinning fraction of 0.45 in the space group P3. The data were collected at the bending magnet beamline X06DA at the Swiss Light Source, which is equipped with the in-house developed multi-axis goniometer PRIGo and the PILATUS 2M detector. A highly redundant 2.2 Å dataset was collected in a number of different crystal orientations. A substructure solution could only be obtained after 50000 SHELXD [3] tries. Automatic model building after phasing and density modification resulted in a model with the majority of residues built correctly. We will present this particularly difficult case together with other more routine cases, all solved with the same experimental setup and at the beamline X06DA.