A rational engineering strategy for designing protein a-binding camelid single-domain antibodies

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed

SARS Coronavirus nsp1 Protein Induces Template-Dependent Endonucleolytic Cleavage of mRNAs: Viral mRNAs Are Resistant to nsp1-Induced RNA Cleavage

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5′ untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection

Solvation models: Theory and validation

Water plays an active role in many fundamental phenomena in cellular systems such as molecular recognition, folding and conformational equilibria, reaction kinetics and phase partitioning. Hence, our ability to account for the energetics of these processes is highly dependent on the models we use for calculating solvation effects. For example, theoretical prediction of protein-ligand binding modes (i.e., docking) and binding affinities (i.e., scoring) requires an accurate description of the change in hydration that accompanies solute binding. In this review, we discuss the challenges of constructing solvation models that capture these effects, with an emphasis on continuum models and on more recent developments in the field. In our discussion of methods, relatively greater attention will be given to boundary element solutions to the Poisson equation and to nonpolar solvation models, two areas that have become increasingly important but are likely to be less familiar to many readers. The other focus will be upon the trending efforts for evaluating solvation models in order to uncover limitations, biases, and potentially attractive directions for their improvement and applicability. The prospective and retrospective performance of a variety of solvation models in the SAMPL blind challenges will be discussed in detail. After just a few years, these benchmarking exercises have already had a tangible effect in guiding the improvement of solvation models. \ua9 2014 Bentham Science Publishers.Peer reviewed: YesNRC publication: Ye

Cathepsin X

NRC publication: Ye

Calculation of ionic bridge contributions to homospecific interactions mediated by proteoglycans

NRC publication: Ye

Cathepsin X

NRC publication: Ye

Exhaustive docking and solvated interaction energy scoring: lessons learned from the SAMPL4 challenge

We continued prospective assessments of the Wilma-solvated interaction energy (SIE) platform for pose prediction, binding affinity prediction, and virtual screening on the challenging SAMPL4 data sets including the HIV-integrase inhibitor and two host-guest systems. New features of the docking algorithm and scoring function are tested here prospectively for the first time. Wilma-SIE provides good correlations with actual binding affinities over a wide range of binding affinities that includes strong binders as in the case of SAMPL4 host-guest systems. Absolute binding affinities are also reproduced with appropriate training of the scoring function on available data sets or from comparative estimation of the change in target's vibrational entropy. Even when binding modes are known, SIE predictions lack correlation with experimental affinities within dynamic ranges below 2\ua0kcal/mol as in the case of HIV-integrase ligands, but they correctly signaled the narrowness of the dynamic range. Using a common protein structure for all ligands can reduce the noise, while incorporating a more sophisticated solvation treatment improves absolute predictions. The HIV-integrase virtual screening data set consists of promiscuous weak binders with relatively high flexibility and thus it falls outside of the applicability domain of the Wilma-SIE docking platform. Despite these difficulties, unbiased docking around three known binding sites of the enzyme resulted in over a third of ligands being docked within 2\ua0\uc5 from their actual poses and over half of the ligands docked in the correct site, leading to better-than-random virtual screening results. \ua9 2014 Her Majesty the Queen in Right of Canada.Peer reviewed: YesNRC publication: Ye