Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
Abstract
Herein, we systematically and quantitatively evaluate whether endoplasmic reticulum (ER) proteostasis factors impact the mutational tolerance of secretory pathway proteins. We focus on influenza hemagluttinin (HA), a viral membrane protein that folds in the host's ER via a complex pathway. By integrating chemical methods to modulate ER proteostasis with deep mutational scanning to assess mutational tolerance, we discover that upregulation of ER proteostasis factors broadly enhances HA mutational tolerance across diverse structural elements. Remarkably, this proteostasis network-enhanced mutational tolerance occurs at the same sites where mutational tolerance is most reduced by propagation at fever-like temperature. These findings have important implications for influenza evolution, because influenza immune escape is contingent on HA possessing sufficient mutational tolerance to evade antibodies while maintaining the capacity to fold and function. More broadly, this work provides the first experimental evidence that ER proteostasis mechanisms define the mutational tolerance and, therefore, the evolution of secretory pathway proteins.
Data availability
FASTQ files for DMS sequencing are available in the Sequence Read Archive under accession number SRP149672. The deep mutational scanning data analysis will be available upon publication at https://github.com/amphilli/HA_DMS_2018, and is also available in Dataset 1. All differential selection values from deep mutational scanning (pre- and post-filtering) are available in Figure 5-source data 1. The complete RNAseq data are available from GEO under accession number GSE115168.
-
ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutininPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE115168).
Article and author information
Author details
Funding
National Science Foundation (CAREER Award 1652390)
- Matthew D Shoulders
National Science Foundation (Graduate Research Fellowship)
- Angela M Phillips
Richard and Susan Smith Family Foundation (Award for Excellence in Biomedical Research)
- Matthew D Shoulders
Massachusetts Institute of Technology
- Matthew D Shoulders
Tufts University
- Yu-Shan Lin
National Cancer Institute (Koch Institute Support (core) Grant P30-CA14051)
- Vincent L Butty
- Matthew D Shoulders
National Institutes of Health (Al127893)
- Jesse D Bloom
Howard Hughes Medical Institute (Faculty Scholars grant)
- Jesse D Bloom
Simons Foundation (Faculty Scholars grant)
- Jesse D Bloom
National Institute of Environmental Health Sciences (MIT Center for Environmental Health Sciences (core) Grant P30-ES002109)
- Vincent L Butty
- Matthew D Shoulders
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Franz-Ulrich Hartl, Max Planck Institute for Biochemistry, Germany
Version history
- Received: June 6, 2018
- Accepted: August 29, 2018
- Accepted Manuscript published: September 6, 2018 (version 1)
- Version of Record published: October 4, 2018 (version 2)
Copyright
© 2018, Phillips et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,426
- Page views
-
- 391
- Downloads
-
- 19
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.
-
- Biochemistry and Chemical Biology
Organizations that fund research are keen to ensure that their grant selection processes are fair and equitable for all applicants. In 2020, the Arnold and Mabel Beckman Foundation introduced blinding to the first stage of the process used to review applications for Beckman Young Investigator (BYI) awards: applicants were instructed to blind the technical proposal in their initial Letter of Intent by omitting their name, gender, gender-identifying pronouns, and institutional information. Here we examine the impact of this change by comparing the data on gender and institutional prestige of the applicants in the first four years of the new policy (BYI award years 2021–2024) with data on the last four years of the old policy (2017–2020). We find that under the new policy, the distribution of applicants invited to submit a full application shifted from those affiliated with institutions regarded as more prestigious to those outside of this group, and that this trend continued through to the final program awards. We did not find evidence of a shift in the distribution of applicants with respect to gender.