About

S. Walter Englander, Ph.D, is an Emeritus Professor of Biochemistry and Biophysics at the University of Pennsylvania's Perelman School of Medicine. His laboratory specializes in studying macromolecular structure, dynamics, and function, with a focus on protein and nucleic acid research using hydrogen exchange (HX) approaches. His work has explained the chemistry of HX processes in proteins and nucleic acids and formulated physical models to understand how internal motions influence HX rates. Englander's research involves developing and applying specialized hydrogen exchange methods to measure specific parts of proteins involved in various functions, including protein folding on a sub-second time scale and energetic stability of bonding interactions. His laboratory employs biophysical techniques such as 2D NMR and mutational analysis, and his work has contributed to a coherent explanation of protein folding mechanisms.

Research topics

• Chemistry
• Cell biology
• Biology
• Biochemistry

Selected publications

• HX and Me: Understanding Allostery, Folding, and Protein Machines

  Annual Review of Biophysics · 2023-01-11 · 12 citations

  reviewOpen access1st authorCorresponding

  My accidental encounter with protein hydrogen exchange (HX) at its very beginning and its continued development through my scientific career have led us to a series of advances in HX measurement, interpretation, and cutting edge biophysical applications. After some thoughts about how life brought me there, I take the opportunity to reflect on our early studies of allosteric structure and energy change in hemoglobin, the still-current protein folding problem, and our most recent forward-looking studies on protein machines.

  PublisherOA PDFDOI

• Comparison of the structure-function properties of wild-type human apoA-V and a C-terminal truncation associated with elevated plasma triglycerides

  medRxiv · 2023-02-23 · 1 citations

  preprintOpen access

  Abstract Background Plasma triglycerides (TGs) are causally associated with coronary artery disease and acute pancreatitis. Apolipoprotein A-V (apoA-V, gene APOA5 ) is a liver-secreted protein that is carried on triglyceride-rich lipoproteins and promotes the enzymatic activity of lipoprotein lipase (LPL), thereby reducing TG levels. Little is known about apoA-V structure-function; naturally occurring human APOA5 variants can provide novel insights. Methods We used hydrogen-deuterium exchange mass spectrometry to determine the secondary structure of human apoA-V in lipid-free and lipid-associated conditions and identified a C-terminal hydrophobic face. Then, we used genomic data in the Penn Medicine Biobank to identify a rare variant, Q252X, predicted to specifically eliminate this region. We interrogated the function of apoA-V Q252X using recombinant protein in vitro and in vivo in apoa5 knockout mice. Results Human apoA-V Q252X carriers exhibited elevated plasma TG levels consistent with loss of function. Apoa5 knockout mice injected with AAV vectors expressing wildtype and variant APOA5 -AAV recapitulated this phenotype. Part of the loss of function is due to reduced mRNA expression. Functionally, recombinant apoA-V Q252X was more readily soluble in aqueous solutions and more exchangeable with lipoproteins than WT apoA-V. Despite lacking the C- terminal hydrophobic region (a putative lipid binding domain) this protein also decreased plasma TG in vivo . Conclusions Deletion of apoA-V’s C-terminus leads to reduced apoA-V bioavailability in vivo and higher TG levels. However, the C-terminus is not required for lipoprotein binding or enhancement of intravascular lipolytic activity. WT apoA-V is highly prone to aggregation, and this property is markedly reduced in recombinant apoA-V lacking the C-terminus.

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• Correction: A conserved strategy for structure change and energy transduction in Hsp104 and other AAA+ protein motors

  Journal of Biological Chemistry · 2021-10-13

  erratumOpen accessSenior author

  The above review included an error on the top of page six under the heading “Optical tweezers burst and dwell phases represent the closed/SPr and open/NEx states.” The word “open” was used when “closed” was intended. The corrected sentence should read: “The OT observation that substrate translocation occurs in the burst phase associates the OT kinetic burst phase with the cryo-EM structural closed state.”

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• ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes

  Cell Reports · 2021 · 26 citations

  • Cell biology
  • Biology
  • Biochemistry

  WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. Our crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also find that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, our structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.

  PublisherDOI

• Abstract 121: Exploiting Natural Genetic Variation In The Human Triglyceride Regulator <i>APOA5</i> To Understand Its Function

  Arteriosclerosis Thrombosis and Vascular Biology · 2021-09-01 · 1 citations

  article

  Plasma triglycerides (TGs) are an independent predictor of the risk for CAD, the leading cause of death worldwide. TGs are also positively associated with risk and severity of hyperTG-induced acute pancreatitis (HTG-AP). Current therapies are often insufficient in reducing extremely elevated TGs. We believe that apolipoprotein A-V (apoA-V, encoded by APOA5) can fill this unmet medical need. ApoA-V is a potent modulator of TG metabolism; it enhances lipoprotein lipase TG hydrolysis. We hypothesize that naturally occurring human APOA5 variants can inform ApoA-V function and identify novel ApoA-V based therapeutic axes. We used the Penn Medicine Biobank (PMBB) to identify and measure plasma TGs of carriers of APOA5 variants predicted to change ApoA-V structure-function. Then, we used hydrogen-deuterium exchange mass spectroscopy to determine the secondary structure of ApoA-V, thereby identifying putative functional domains onto which we mapped our variants of interest. Finally, we characterized the plasma lipid effects of these mutants using adeno-associated viral (AAV) vectors in apoa5 knockout (KO) mice. We identified APOA5 variants associated with changes in plasma TGs. These variants primarily fall near the central heparin binding domain or C-terminal lipid binding domain. We selected APOA5 Q275X, which removes the entire lipid binding domain, for further interrogation. Apoa5 KO mice that received APOA5 Q275X AAV had higher plasma TGs than mice treated with WT APOA5 AAV. While WT ApoA-V protein associated with VLDL and HDL particles, Q275X ApoA-V protein appeared in lipoprotein free fractions. We have identified APOA5 variants associated with plasma TG phenotypes in humans, and mapped them to an experimentally determined ApoA-V secondary structure to identify the functional domains likely impacted. We have identified APOA5 Q275X as a loss of function variant that fails to bind lipoprotein particles and is associated with elevated plasma TGs. Continued study of this and other interesting naturally occurring variants will provide insight into the function of ApoA-V in TG metabolism. These insights can help us to therapeutically enhance ApoA-V to rapidly reduce TG levels during acute HTG-AP and to help prevent recurrent HTG-AP.

  PublisherDOI

• A conserved strategy for structure change and energy transduction in Hsp104 and other AAA+ protein motors

  Journal of Biological Chemistry · 2021-08-09 · 5 citations

  reviewOpen accessSenior authorCorresponding

  The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input "open" state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured "closed" state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.

  PublisherOA PDFDOI

• ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes

  bioRxiv (Cold Spring Harbor Laboratory) · 2021-04-30 · 2 citations

  preprintOpen access

  SUMMARY WNTs play key roles in development and disease, by binding both Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors that include the ROR and RYK receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2). RORs bind WNTs though a FZD-related cysteine-rich domain (CRD), and RYKs through a WNT-inhibitory factor (WIF) domain. Our structures suggest that neither the Nrk CRD nor the Drl-2 WIF domain can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable with a WNT acyl chain. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also show that DWnt-5, which regulates Drosophila ROR and RYK orthologs, lacks an acyl chain. Together with analysis of WNT/receptor interaction sites, these structures provide new insight into how WNTs recruit their RTK co-receptors into signaling complexes.

  PublisherOA PDFDOI

• Characterization of small molecule induced changes in Parkinson’s-related trafficking via the Nedd4 ubiquitin signaling cascade

  bioRxiv (Cold Spring Harbor Laboratory) · 2020-06-02 · 1 citations

  preprintOpen access

  Summary The benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson’s disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (K D app = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro . Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its colocalization. Ubiquitin-enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking and transport associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism.

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• ApoC-III helical structure determines its ability to bind plasma lipoproteins and inhibit Lipoprotein Lipase-mediated triglyceride lipolysis

  bioRxiv (Cold Spring Harbor Laboratory) · 2020-07-03 · 1 citations

  preprintOpen access

  Abstract In humans, apolipoprotein C-III (apoC-III) plasma levels have been associated with increased risk of cardiovascular disease. This association is in part explained by the effects of apoC-III on triglyceride (TG) metabolism; apoC-III raises plasma TG by increasing very low density lipoprotein (VLDL) secretion, inhibiting lipoprotein lipase (LPL)-mediated TG lipolysis, and impairing the removal of triglyceride-rich lipoprotein (TRL) remnants from the circulation. In this study, we explored the structure-function relationship the interaction of apoC-III with plasma lipoproteins and its ultimate impact on LPL activity. The structural and functional properties of wild-type (WT) apoC-III were compared with two missense variants previously associated with lower (A23T) and higher (Q38K) plasma TG. ApoC-III in the lipid-free state is unstructured but its helix content and stability increases when bound to lipid. Lipid-bound apoC-III contains two alpha helices spanning residues amino acids 11 - 38 (helix 1) and 44 – 64 (helix 2). Investigation of the structural and functional consequences of the A23T and Q38K variants showed that these amino acid substitutions within helix 1 do not significantly alter the stability of the helical structure but affect its hydrophilic-lipophilic properties. The A23T substitution impairs lipoprotein binding capacity, reduces LPL inhibition, and ultimately leads to lower plasma TG levels. Conversely, the Q to K substitution at position 38 enhances the lipid affinity of helix 1, increases TRL binding capacity and LPL inhibition, and is associated with hypertriglyceridemia. This study indicates that structural modifications that perturb the hydrophilic/lipophilic properties of the alpha helices can modulate the hypertriglyceridemic effects of apoC-III.

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• Structural and kinetic basis for the regulation and potentiation of Hsp104 function

  Proceedings of the National Academy of Sciences · 2020-04-10 · 32 citations

  articleOpen accessSenior authorCorresponding

  Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM075105

  NIH · $951k · 2010

• NIH Grant R01DK011295

  NIH · $2.4M · 2000

• Protein structure and function by hydrogen exchange mass spectrometry analysis

  NIH · $10.0M · 1983–2019

• How do proteins fold: Mechanism and principles

  NSF · $600k · 2019–2023

• Protein structure and function by hydrogen exchange mass spectrometry analysis

  NIH · $323k · 1983–2018

Frequent coauthors

• Leland Mayne

  University of Pennsylvania

  188 shared

• Krishna M.G. Mallela

  University of Colorado Anschutz Medical Campus

  51 shared

• Jon N. Rumbley

  University of Minnesota, Duluth

  38 shared

• Zhong-Yuan Kan

  Providence College

  38 shared

• Tobin R. Sosnick

  University of Chicago

  36 shared

• Joan J. Englander

  University of Pennsylvania

  33 shared

• Yawen Bai

  National Institutes of Health

  27 shared

• Haripada Maity

  Eli Lilly (United States)

  25 shared

Labs

• S. Walter Englander LaboratoryPI

Education

• MS, PhD, Biophysics

  University of Pittsburgh

  1960