Flexibility and mobility of SARS-CoV-2-related protein structures.

The worldwide CoVid-19 pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as possible. Existing academic, governmental and industrial institutions and companies have engaged in large-scale screening of existing drugs, in vitro, in vivo and in silico. Here, we are using in silico modelling of possible SARS-CoV-2 drug targets, as deposited on the Protein Databank (PDB), and ascertain their dynamics, flexibility and rigidity. For example, for the SARS-CoV-2 spike protein-using its complete homo-trimer configuration with 2905 residues-our method identifies a large-scale opening and closing of the S1 subunit through movement of the S[Formula: see text] domain. We compute the full structural information of this process, allowing for docking studies with possible drug structures. In a dedicated database, we present similarly detailed results for the further, nearly 300, thus far resolved SARS-CoV-2-related protein structures in the PDB.

'Something in the way she moves': The functional significance of flexibility in the multiple roles of protein disulfide isomerase (PDI).

Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI.

Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome.

BACKGROUND: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca(2+)-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly(204)Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. RESULTS: In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca(2+) binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca(2+) during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. CONCLUSIONS: We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca(2+) binding during biosynthesis.

Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10(-5) M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding - differential affinity, rapid ligand exchange and conformational flexibility.

High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility.

ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxin-fold domains homologous with the non-catalytic b and b' domains of PDI. The structure in solution of the N-terminal b-like domain of ERp27 was solved using high-resolution NMR data. The structure confirms that it has the thioredoxin fold and that ERp27 is a member of the PDI family. (15)N-NMR relaxation data were obtained and ModelFree analysis highlighted limited exchange contributions and slow internal motions, and indicated that the domain has an average order parameter S(2) of 0.79. Comparison of the single-domain structure determined in the present study with the equivalent domain within full-length ERp27, determined independently by X-ray diffraction, indicated very close agreement. The domain interface inferred from NMR data in solution was much more extensive than that observed in the X-ray structure, suggesting that the domains flex independently and that crystallization selects one specific interdomain orientation. This led us to apply a new rapid method to simulate the flexibility of the full-length protein, establishing that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker, consistent with the NMR data.

Assisting oxidative protein folding: how do protein disulphide-isomerases couple conformational and chemical processes in protein folding?

Oxidative folding is the simultaneous process of forming disulphide bonds and native structure in proteins. Pathways of oxidative folding are highly diverse and in eukaryotes are catalysed by protein disulphide isomerases (PDIs). PDI consists of four thioredoxin-like domains, two of which contain active sites responsible for disulphide interchange reactions. The four domains are arranged in a horseshoe shape with the two active sites facing each other at the opening of the horseshoe. An extended hydrophobic surface at the bottom of the horseshoe is responsible for non-covalent, hydrophobic interactions with the folding protein. This binding site is capable of distinguishing between fully-folded and partially- or un-folded proteins. PDI is not only a catalyst of the formation of disulphide bonds, but also catalyses folding steps which involve significant conformational change in the folding protein. This review brings together the latest catalytic and structural data aimed at understanding how this is achieved.

Plasticity of human protein disulfide isomerase: evidence for mobility around the X-linker region and its functional significance.

Protein disulfide isomerase (PDI), which consists of multiple domains arranged as abb'xa'c, is a key enzyme responsible for oxidative folding in the endoplasmic reticulum. In this work we focus on the conformational plasticity of this enzyme. Proteolysis of native human PDI (hPDI) by several proteases consistently targets sites in the C-terminal half of the molecule (x-linker and a' domain) leaving large fragments in which the N terminus is intact. Fluorescence studies on the W111F/W390F mutant of full-length PDI show that its fluorescence is dominated by Trp-347 in the x-linker which acts as an intrinsic reporter and indicates that this linker can move between "capped" and "uncapped" conformations in which it either occupies or exposes the major ligand binding site on the b' domain of hPDI. Studies with a range of constructs and mutants using intrinsic fluorescence, collision quenching, and extrinsic probe fluorescence (1-anilino-8-naphthalene sulfonate) show that the presence of the a' domain in full-length hPDI moderates the ability of the x-linker to generate the capped conformation (compared with shorter fragments) but does not abolish it. Hence, unlike yeast PDI, the major conformational plasticity of full-length hPDI concerns the mobility of the a' domain "arm" relative to the bb' "trunk" mediated by the x-linker. The chaperone and enzymatic activities of these constructs and mutants are consistent with the interpretation that the reversible interaction of the x-linker with the ligand binding site mediates access of protein substrates to this site.

Mapping of the ligand-binding site on the b' domain of human PDI: interaction with peptide ligands and the x-linker region.

PDI (protein disulfide-isomerase) catalyses the formation of native disulfide bonds of secretory proteins in the endoplasmic reticulum. PDI consists of four thioredoxin-like domains, of which two contain redox-active catalytic sites (a and a'), and two do not (b and b'). The b' domain is primarily responsible for substrate binding, although the nature and specificity of the substrate-binding site is still poorly understood. In the present study, we show that the b' domain of human PDI is in conformational exchange, but that its structure is stabilized by the addition of peptide ligands or by binding the x-linker region. The location of the ligand-binding site in b' was mapped by NMR chemical shift perturbation and found to consist primarily of residues from the core beta-sheet and alpha-helices 1 and 3. This site is where the x-linker region binds in the X-ray structure of b'x and we show that peptide ligands can compete with x binding at this site. The finding that x binds in the principal ligand-binding site of b' further supports the hypothesis that x functions to gate access to this site and so modulates PDI activity.