ABSTRACT
Sortase enzymes are cysteine transpeptidases that attach environmental sensors, toxins, and other proteins to the cell surface in Gram-positive bacteria. The recognition motif for many sortases is the cell wall sorting signal (CWSS), LPXTG, where X = any amino acid. Recent work from ourselves and others has described recognition of additional amino acids at a number of positions in the CWSS, specifically at the Thr (or P1) and Gly (or P1') positions. In addition, although standard cleavage occurs between these two residues (P1/P1'), we previously observed that the SrtA enzyme from Streptococcus pneumoniae will cleave after the P1' position when its identity is a Leu or Phe. The stereochemical basis of this alternative cleavage is not known, although homologs, e.g., SrtA from Listeria monocytogenes or Staphylococcus aureus do not show alternative cleavage to a significant extent. Here, we use protein biochemistry, structural biology, and computational biochemistry to predict an alternative binding mode that facilitates alternative cleavage. We use Streptococcus pyogenes SrtA (spySrtA) as our model enzyme, first confirming that it shows similar standard/alternative cleavage ratios for LPATL, LPATF, and LPATY sequences. Molecular dynamics simulations suggest that when P1' is Leu, this amino acid binds in the canonical S1 pocket, pushing the P1 Thr towards solvent. The P4 Leu (L̲PATL) binds as it does in standard binding, resulting in a puckered binding conformation. We use P1 Glu-containing peptides to support our hypotheses, and present the complex structure of spySrtA-LPALA to confirm favorable accommodation of Leu in the S1 pocket. Overall, we structurally characterize an alternative binding mode for spySrtA and specific target sequences, expanding the potential protein engineering possibilities in sortase-mediated ligation applications.
ABSTRACT
Significance: Vaping is an epidemic among young people, but there is little guidance on how medical providers should counsel young adults about vaping. To address this gap, we examined how electronic health record (EHR) systems prompt providers to collect vaping data and interviewed young adults about vaping communications with providers and preferred information sources. Methods: In this mixed methods study, we used survey research methods to explore if prompts exist in EHR systems to guide discussions about vaping with youth seen in primary care. We collected primary care practice information about EHR prompts regarding e-cigarette use from 10 rural North Carolina practices from August 2020 through November 2020 and interviewed 17 young adults (age 18-21 years) who reviewed resources and provided their opinion on the resource's relevance for their age group. Interviews were stratified by vaping status, transcribed, coded, and thematically analyzed. Results: Only 5 of 10 EHR systems included prompts to capture information about vaping and data capture was optional in all 5 cases. Of the 17 interviewees, 10 were female, 14 were White, 3 were non-White and the mean age was 19.6 years. Two central themes emerged. Young adults: 1) were open to confidential, non-confrontational interactions with trusted providers and supported the use of a 2-page resource/discussion guide, questionnaires about vaping, and other waiting room resources, and 2) wanted prevention and cessation resources to be age-appropriate, including medical facts from a trusted source, and to be disseminated via social media platforms used by young adults. Conclusions: We found a lack of EHR functionalities in screening for vaping status hindered patients from receiving counseling on use. Young adults report a willingness to communicate with and learn from trusted providers and to gain understanding from information accessed via social media.
