Mapping Conformational Dynamics to Individual Steps in the TEM-1 β-Lactamase Catalytic Mechanism

Knox, Ruth; Lento, Cristina; Wilson, Derek J.

doi:10.1016/j.jmb.2018.06.045

Cited by 30 publications

(47 citation statements)

References 71 publications

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“…HDX-MS was used to compare the structural dynamics of Pyk2 FERM-kinase with the autonomous FERM or kinase domains. HDX-MS is a useful technique for studying protein folding, protein-protein interactions, allostery, and conformational dynamics [46][47][48][49][50] . HDX-MS measures the rates of backbone amide proton exchange to report on local chemical environments.…”

Section: Resultsmentioning

confidence: 99%

Conformational dynamics of FERM-mediated autoinhibition in Pyk2 tyrosine kinase

Loving

Underbakke

2019

Preprint

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Pyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca 2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and kinase activity profiling. The results reveal FERM-kinase interfaces responsible for autoinhibition. Pyk2 autoinhibition impacts activation loop conformation. In addition, the autoinhibitory FERM-kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.

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Section: Resultsmentioning

confidence: 99%

Conformational dynamics of FERM-mediated autoinhibition in Pyk2 tyrosine kinase

Loving

Underbakke

2019

Preprint

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“…Initial tests on synthetic peptide hormone (bradykinin), neurotransmitter (leucine enkephalin) and consensus anti-freeze protein (CN-AFP) validated the approach by permitting calculation of protection factors, even in natively unstructured regions. The new ms2min system developed here has significant merit from a technical standpoint: considerable potential for HDX-MS resolved at the millisecond level has been demonstrated previously, for example applied to the study of protein folding (11) and to TEM lactamase catalysis (15). The ms2min system stands to be particularly impactful in the future study of time-resolved (i.e.…”

Section: Discussionmentioning

confidence: 95%

“…It has been challenging to provide quantitative rationales for the structural switching that is observed in alternately regulated conformations of enzymes, particularly when the molecular size and complexity are large. One such method that can resolve local (near amino acid) perturbations in stability in arbitrarily large proteins is hydrogen/deuterium-exchange mass spectrometry (HDX-MS) (11)(12)(13)(14)(15)(16). Moreover, these measurements are made in solution without the need to trap specific conformers, which has been used to answer questions of epitope mapping (17), protein-drug binding (18), protein-protein interactions (19), aggregation (20), effects of mutation (12), and allosteric regulation (21).…”

Section: Introductionmentioning

confidence: 99%

Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

Kish

Smith

Subramanian

et al. 2019

Preprint

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Glycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. The sensitivity of these measurements discerned that the 250's loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. Significance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability we provide direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop. Results Fully-automated and flexible pulse-labeling hydrogen/deuterium-exchange mass spectrometry with millisecond precisionOur goal was to quantify the structural switch in solution, along with other coincident dynamic changes in structure between activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of GlyP. To enable this, we developed a fully automated broadband bottom-up HDX-MS approach capable of accurate quantitative assessment of peptide stability. The newly developed ms2min system design ( Figure 1A) offers certain significant advances over the other designs previously employed. Notably, it allows fully-automated, software-selection of mixing times over six orders of magnitude (ms to hours) with 1 ms time resolution, flexible labeling temperature control (0 -25 C), quench temperature control (0 C), on-line connection for 'bottom-up' workflows, two-way communication for reciprocal control of labeling, washing, digestion, desalting and chromatography, automated digestion column wash injection, intercalated blank injections and sample list scheduling of multiple runs. Precision of D-labeling.To evaluate the measurement variability and labeling precision of the system we determined the repeatability of the quench-flow labeling method. On three separate days we measured the D uptake of 50, 70 and 150 replicates of Bradykinin and Leucine enkephalin at 100 ms mixing time. The short labeling period close to the limit of quantification, wa...

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“…The catalytic reaction is followed in real time, with HDX occurring at exposed conformational sites sampled by the working enzyme, after which deuteration is terminated by acid quenching, followed by proteolysis and ESI‐MS analysis of the peptides (Figure A). Among other examples, the Wilson group has recently used this methodology to illustrate specific dynamic modes and changes in enzyme dynamics associated with substrate/inhibitor binding and turnover in the active TEM‐1 β‐lactamase . Their results illustrate that specific dynamic modes experienced by TEM‐1 only occur in presence of certain penicillin substrates or the clavulanate inhibitor.…”

Section: New and Promising Techniques To Investigate Enzyme Dynamicsmentioning

confidence: 99%

“…Differences in deuterium uptake effectively allows extraction and comparison of the conformational landscape sampled by the enzyme under the influence of different ligands along the reaction. Figure adapted from ref . B) Catalytic mechanism of deacylation in TEM‐1 β‐lactamase, whereby Glu166 abstracts a proton from a strictly conserved water molecule to initiate breakdown of the acyl‐enzyme intermediate and regenerate the free enzyme.…”

Section: New and Promising Techniques To Investigate Enzyme Dynamicsmentioning

confidence: 99%