Engineering topics

VanPelt, Jamie

Publications and source records attributed to VanPelt, Jamie.

5 records

DOE OSTI2018

New Delhi Metallo‐Beta‐Lactamase Variants NDM‐4 and NDM‐12 from E. coli Clinical Isolates Exhibit Increased Activity and Stability

Metallo‐Beta‐Lactamases (MBLs) confer resistance to carbapenems, cephalosporins, and penicillins in several clinically relevant Gram‐negative bacteria including Acinetobacter, Pseudomonas aeruginosa, and many Enterobacteriaceae. New Delhi Metallo‐Beta‐Lactamases (NDMs) are amongst some of the most worrisome and prevalent MBLs. This family of MBLs is capable of hydrolyzing all generations of bicyclic beta‐lactams. Two new NDM variants, NDM‐4 and ‐12, have recently been identified in E. coli clinical isolates. NDM‐4 contains an M154L substitution while NDM‐12 bears both M154L and G222D mutations. Considering the clinical significance of Gram‐negative species possessing these enzymes, there is a need to characterize the structures and activities of these variants in order to provide a better basis for drug discovery efforts. We have found that NDM‐4 and NDM‐12 possess higher hydrolytic activity compared to NDM‐1. Our differential scanning fluorimetry studies suggest that the M154L mutation confers increased thermal stability and improved affinity for active site catalytic Zn metal ions. The additional G222D mutation of NDM‐12 further improves stability and Zn affinity. M154L, found in the secondary coordination sphere, accounts for changes in stability by acting as a buttress to the loop containing His 122 , a direct Zn coordinating residue. G222D, on the other hand, is a mutation located in a loop just above the active site approximately 10 Å from the active site zinc ions. Together, our crystal structures of these NDM variants, including the first known structure of NDM‐12, biochemical analyses, and biophysical analyses provide a basis for understanding the roles of key residues surrounding the active site within NDM variants. A better understanding of clinically relevant NDM variants is essential in the fight against MBL‐mediated antibiotic resistance. Our structural, biochemical, and biophysical studies will help to inform drug design efforts to develop new MBL inhibitors. Support or Funding Information The authors acknowledge financial support from the US National Institutes of Health (Award No. R01 GM111926 to RCP, DLT, MWC, and WF) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Williams, Cameron↗

DOE OSTI2018

Shifting Sands: Modulating Interactions and Outputs of the CHIP‐Hsp70/HOP‐Hsp70 Protein Quality Control Complexes

The 70‐kilodalton heat shock protein (Hsp70) interacts with tetratricopeptide repeat (TPR) domains of CHIP (C‐terminus of Hsp70 Interacting Protein) and HOP (Hsp Organizing Protein) using a two‐carboxylate clamp binding mechanism. Differential affinity for the TPR domains of CHIP and HOP are achieved through interactions with the six residues preceding the carboxy‐terminal Asp 641 of Hsp70. In particular, phosphorylation of Hsp70 Thr 636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Given that CHIP‐Hsp70 complexes promote ubiquitin‐mediated degradation of Hsp70‐chaperoned clients while HOP‐Hsp70 complexes promote refolding of Hsp70‐chaperoned clients, phosphorylation of Hsp70 Thr 636 acts as a switch that directs the output and function of cellular protein quality control. Herein, through biophysical assays and new structures of CHIP‐Hsp70 and Hop‐Hsp70 we identify the molecular mechanism that enables phosphorylation‐driven switching that directs interactions within the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes. We also present a structure‐guided mutation of Hsp70 that bypasses Thr 636 phosphorylation‐driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP. This structure‐guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes toward chaperoned ubiquitination via the CHIP‐Hsp70 complex. Support or Funding Information The authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Page, Richard C.↗

DOE OSTI2018

Interactions and Dynamics Within CHIP/Hsp70 Complexes

Interactions between the E3 ubiquitin ligase CHIP (C‐terminus of Hsp70 interacting protein) and the chaperone Hsp70 represent the physical intersection of two competing protein quality control efforts: refolding, and degradation. Interaction with CHIP allows for the formation of a productive encounter complex that facilitates ubiquitination of Hsp70‐bound substrates, thereby targeting those proteins for proteasomal degradation. Central to the CHIP/Hsp70 interaction are changes in structure and dynamics that occur upon binding. Although recruitment of Hsp70 to CHIP via interaction with the CHIP tetratricopeptide repeat (TPR) domain is well established, alterations in structure and dynamics of CHIP and Hsp70 upon binding are not well understood. Using NMR we describe residue‐level changes in dynamics to the CHIP‐TPR domain that occur upon binding Hsp70. We also utilize small angle X‐ray scattering (SAXS) and electron paramagnetic resonance (EPR) to identify motions with the CHIP/Hsp70 complex. Independent SAXS and EPR experiments both point to a mobile Hsp70 substrate binding domain that is only partially constrained through interactions with the CHIP‐TPR. Building upon static models of the full‐length CHIP/Hsp70 complex, afforded by our recent crystal structure of the CHIP‐TPR/Hsp70‐lid‐tail complex, we have assembled a dynamic model of the CHIP‐Hsp70 complex. This dynamic model sheds light on the function of the CHIP/Hsp70 chaperoned ubiquitination complex and how CHIP harnesses dynamics of Hsp70 to overcome significant distance separations identified in earlier static models of the CHIP/Hsp70 complex. Support or Funding Information The authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Mannion, Matthew M.↗

DOE OSTI2018

A Noncanonical Metal Center Drives the Activity of the Sediminispirochaeta smaragdinae Metallo-β-lactamase SPS-1

In an effort to evaluate whether a recently reported putative metallo-β-lactamase (MβL) contains a novel MβL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not β-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn 1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn 2 site that is similar to that in NDM-1 and other subclass B1 MβLs; however, the Zn 1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn 1 metal is displaced nearly 1 Å from the position reported in other MβLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of β-lactam antibiotics. Here, this study reveals a novel metal binding site in MβLs and suggests that the targeting of metal binding sites in MβLs with inhibitors is now more challenging with the identification of this new MβL.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving To Overcome Zinc Scarcity

Use and misuse of antibiotics have driven the evolution of serine β-lactamases to better recognize new generations of β-lactam drugs, but the selective pressures driving evolution of metallo-β-lactamases are less clear. Here in this paper, we present evidence that New Delhi metallo-β-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt(II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. Lastly, these studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

59 BASIC BIOLOGICAL SCIENCES↗