Sofiane Saouane

Protein function depends on their conformational flexibility. Complementary to temperature-dependent studies, high-pressure (HP) macromolecular crystallography is well suited to exploring the conformational space of proteins at atomic resolution. We have developed a new HP cell concept for macromolecular X-ray crystallography that overcomes the limitations of current devices. It consists of a single-crystalline sapphire capillary, which is loaded with multiple crystals and pressurized with helium at freely adjustable gas pressures of up to 120 MPa. Studying five different proteins, we generally observed a compression of the protein structures and a decrease in thermal displacement parameters at high pressure. Structurally, observations included the compression of a central tunnel of a biotechnologically relevant enzyme and the stabilization of ligand binding in the membrane protein photosynthetic reaction center of Cereibacter sphaeroides . For two pharmaceutically relevant proteins, the human kinase DRAK2 and SARS-CoV-2 main protease, we identified concerted movements of subdomains. Such knowledge is essential for predictions of protein dynamics and defined conformational states, offering the potential for improved structure-based drug development.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host’s innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

A large-scale screen to target SARS-CoV-2: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is initially expressed as two large polyproteins. Its main protease, M ^pro , is essential to yield functional viral proteins, making it a key drug target. Günther et al. used x-ray crystallography to screen more than 5000 compounds that are either approved drugs or drugs in clinical trials. The screen identified 37 compounds that bind to M ^pro . High-resolution structures showed that most compounds bind at the active site but also revealed two allosteric sites where binding of a drug causes conformational changes that affect the active site. In cell-based assays, seven compounds had antiviral activity without toxicity. The most potent, calpeptin, binds covalently in the active site, whereas the second most potent, pelitinib, binds at an allosteric site. Science , this issue p. 642

This paper reports a new packing type of α-cyclodextrin inclusion complexes, obtained here with succinic acid under low-temperature crystallization conditions. The structure of the 1:1 complex is characterized by heavy disorder of the guest, the solvent, and part of the host. The crystal packing belongs to the known channel-type structure; the basic structural unit is composed of cyclodextrin trimers, as opposed to the known isolated molecular or dimeric constructs, packed along the c-axis. Each trimer is made of crystallographically independent molecules assembled in a stacked vase-like cluster. A multi-temperature single-crystal X-ray diffraction analysis reveals the presence of dynamic disorder.

The X-ray single-crystal structure of (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, commonly known as (+)-6-aminopenicillanic acid (C₈H₁₂N₂O₃S) and a precursor of a variety of semi-synthetic penicillins, has been determined from synchrotron data at 150 K. The structure represents an ordered zwitterion and the crystals are nonmerohedrally twinned. The crystal structure is composed of a three-dimensional network built by three charge-assisted hydrogen bonds between the ammonium and carboxylate groups. The complementary analysis of the crystal packing by thePIXELmethod brings to light the nature and ranking of the energetically most stabilizing intermolecular interaction energies. In accordance with the zwitterionic nature of the structure,PIXELlattice energy calculations confirm the predominance of the Coulombic term (−379.1 kJ mol⁻¹) ahead of the polarization (−141.4 kJ mol⁻¹), dispersion (−133.7 kJ mol⁻¹) and repulsion (266.3 kJ mol⁻¹) contributions.

The solid-state polymorphism of the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF₆], has been investigated via low-temperature and high-pressure crystallisation experiments. The samples have been characterised by single-crystal X-ray diffraction, optical microscopy and Raman spectroscopy. The solid-state phase behaviour of the compound is confirmed and clarified with respect to previous phase diagrams. The structures of the previously reported γ-form, which essentially exhibits a G′T cation conformation, as well as those of the elusive β- and α-forms, are reported. Crystals of the β-phase are twinned and the structure is heavily disordered; the cation conformation in this form is predominantly TT, though significant contributions from other less frequently encountered conformers are also observed at low temperature and high pressure. The cation conformation in the α-form is GT; the presence of the G′T conformer at 193 K in this phase can be eliminated on cooling to 100 K. Whilst X-ray structural data are overall in good agreement with previous interpretations based on Raman and NMR studies, they also reveal a more subtle interplay of intermolecular interactions, which give rise to a wider range of conformers than previously considered.

The technique of high-pressure crystallisation from solution has been applied to the study of α-cyclodextrin hydrate formation. A previously unobserved hydrate, here termed Ib, has been obtained at 0.65 GPa and characterised by single-crystal X-ray diffraction using synchrotron radiation. This form exhibits a combination of structural features of other known hydrates and hydration states obtained by theoretical calculations but never found experimentally before.