Solution structure and electrostatic properties of an SH2 domain/phosphopeptide complex

grantor: University of TorontoSH2 domains are small (~100 amino acid) protein recognition domains found in numerous proteins involved in signal transduction which bind to sites of tyrosine phosphorylation with high affinity in a sequence-dependent manner. We have focused on the SH2 domains of phospholipase C-ã (PLC-ã), which provide a link between activated growth factor receptors via binding through its two SH2 domains and the production of the second messengers IP3 and DAG. The interaction of PLC-ã with the platelet derived growth factor receptor (PDGFR) is at sequences about Tyr 1021 of the PDGFR, and disruption of this interaction results in decreased cell growth following growth factor stimulation. Binding studies using degenerate phosphopeptide libraries suggest that this interaction involves the C-terminal (PLCC), and not N-terminal (PLCN) SH2 domain of PLC-ã. Thus we have studied this interaction involving the PLCC SH2 domain and a 12 amino acid phosphopeptide representing sequences about Tyr 1021 using heteronuclear NMR techniques. I was involved in the cloning and purification of this SH2 domain and preparation of NMR samples of this protein/peptide complex. A full structural determination was performed on this complex in collaboration with Dr. Steve Pascal. During structure determination, I defined the conformation of the phosphopeptide in this complex, as well as demonstrating protein-peptide contacts. Protein/peptide NOEs involving pTyr resonances defined a large positively-charged pocket containing four arginine residues which bound this residue. NOEs could not define contacts with the pTyr phosphate group, and we used large downfield chemical shift changes of guanidinium group resonances to do so. pH titration studies demonstrated that the pTyr phosphate group is bound in the -2 charge state with several residues held in place to facilitate pTyr binding by a complex hydrogen bonding network. A large hydrophobic cavity on the SH2 domain surface bound six residues C-terminal to pTyr, and in particular, the Ile +1 and Pro +3 residues were deeply buried. Thus a combination of NMR techniques involving NMR assignment, structure determination and pH titration studies provided significant insights into the specific binding of SH2 domains.Ph.D

Risk of COVID-19 after natural infection or vaccinationResearch in context

Summary: Background: While vaccines have established utility against COVID-19, phase 3 efficacy studies have generally not comprehensively evaluated protection provided by previous infection or hybrid immunity (previous infection plus vaccination). Individual patient data from US government-supported harmonized vaccine trials provide an unprecedented sample population to address this issue. We characterized the protective efficacy of previous SARS-CoV-2 infection and hybrid immunity against COVID-19 early in the pandemic over three-to six-month follow-up and compared with vaccine-associated protection. Methods: In this post-hoc cross-protocol analysis of the Moderna, AstraZeneca, Janssen, and Novavax COVID-19 vaccine clinical trials, we allocated participants into four groups based on previous-infection status at enrolment and treatment: no previous infection/placebo; previous infection/placebo; no previous infection/vaccine; and previous infection/vaccine. The main outcome was RT-PCR-confirmed COVID-19 >7–15 days (per original protocols) after final study injection. We calculated crude and adjusted efficacy measures. Findings: Previous infection/placebo participants had a 92% decreased risk of future COVID-19 compared to no previous infection/placebo participants (overall hazard ratio [HR] ratio: 0.08; 95% CI: 0.05–0.13). Among single-dose Janssen participants, hybrid immunity conferred greater protection than vaccine alone (HR: 0.03; 95% CI: 0.01–0.10). Too few infections were observed to draw statistical inferences comparing hybrid immunity to vaccine alone for other trials. Vaccination, previous infection, and hybrid immunity all provided near-complete protection against severe disease. Interpretation: Previous infection, any hybrid immunity, and two-dose vaccination all provided substantial protection against symptomatic and severe COVID-19 through the early Delta period. Thus, as a surrogate for natural infection, vaccination remains the safest approach to protection. Funding: National Institutes of Health