Conformational selection and induced changes along the catalytic cycle of <i>Escherichia coli</i> dihydrofolate reductase

Weikl, Thomas R.; Boehr, David D.

doi:10.1002/prot.24123

Cited by 23 publications

(45 citation statements)

References 76 publications

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“…It has been noted that in prokaryotes (such as E. coli) the concentrations of NADPH and NADP + are similar, whereas in eukaryotic cells the concentration of NADP + is typically 100 times smaller than the NADPH concentration (33). Therefore, a large K p value in E. coli would significantly stall the DHFR catalytic cycle through a greater degree of product inhibition, where…”

Section: Resultsmentioning

confidence: 99%

“…It has been shown that the binding of inhibitors, such as TMP to ecDHFR, can affect the conformational states of the protein (34). Because the flexible PEKN domain resides over the TMP binding pocket [Protein Data Bank (PDB) ID2W3A] (15), the local conformational fluctuations in this region should affect the binding and dissociation of ligands (17,33,35). Again, within experimental errors, all PEKNcontaining ecDHFR variants show the same binding affinity for TMP, and neither the L28F nor the N23PP mutations exhibit any additional impacts on TMP binding.…”

Section: Resultsmentioning

confidence: 99%

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Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Liu

Hanoian

French

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

141

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With the rapidly growing wealth of genomic data, experimental inquiries on the functional significance of important divergence sites in protein evolution are becoming more accessible. Here we trace the evolution of dihydrofolate reductase (DHFR) and identify multiple key divergence sites among 233 species between humans and bacteria. We connect these sites, experimentally and computationally, to changes in the enzyme's binding properties and catalytic efficiency. One of the identified evolutionarily important sites is the N23PP modification (∼mid-Devonian, 415-385 Mya), which alters the conformational states of the active site loop in Escherichia coli dihydrofolate reductase and negatively impacts catalysis. This enzyme activity was restored with the inclusion of an evolutionarily significant lid domain (G51PEKN in E. coli enzyme; ∼2.4 Gya). Guided by this evolutionary genomic analysis, we generated a human-like E. coli dihydrofolate reductase variant through three simple mutations despite only 26% sequence identity between native human and E. coli DHFRs. Molecular dynamics simulations indicate that the overall conformational motions of the protein within a common scaffold are retained throughout evolution, although subtle changes to the equilibrium conformational sampling altered the free energy barrier of the enzymatic reaction in some cases. The data presented here provide a glimpse into the evolutionary trajectory of functional DHFR through its protein sequence space that lead to the diverged binding and catalytic properties of the E. coli and human enzymes.phylogenetic | EVB

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Liu

Hanoian

French

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

141

View full text Add to dashboard Cite

show abstract

“…The loop interactions in DHFR are also likely important for catalytic flux, which may depend on the cellular environment and NADP + /NADPH concentrations . For example, the occluded conformation is disfavored by a polyproline sequence at the C‐terminal end of the Met20 loop in human DHFR.…”

Section: Modulation Of Loop Dynamicsmentioning

confidence: 99%

Engineered control of enzyme structural dynamics and function

2018

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Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine.

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“…The temporal ordering of the conformational changes and binding events of the pathway requires transition times for ligand binding and unbinding that are small compared to the dwell times in the states P 2 and P 2 L (see text). The effective on‐ and off‐rate constants of the pathway are

k_{on} ≃ u_{12} k_{2}^{+} b_{23} / (u_{21} k_{2}^{-} + u_{21} b_{23} + k_{2}^{+} [L] b_{23})

and

k_{off} ≃ u_{21} k_{2}^{-} b_{32} / (u_{21} k_{2}^{-} + u_{21} b_{23} + k_{2}^{+} [L] b_{23})

…”

Section: Conformational Selection and Induced Changes Are Two Sides Omentioning

confidence: 99%

“…Under “pseudo‐first order” conditions with a ligand concentration

[L]

that greatly exceeds the protein concentration, the relaxation process of the two‐step binding mechanisms in Figure (a,b) is a bi‐exponential process . The slower, dominant rate of this process has the general from

k_{obs} = k_{1} = \frac{1}{2} true(σ - \sqrt{σ^{2} - 4 ρ} true)

while the faster rate is

k_{2} = \frac{1}{2} true(σ + \sqrt{σ^{2} - 4 ρ} true)

.…”

Section: Effective Overall Rates Of Conformational‐selection and Indmentioning

confidence: 99%

Conformational selection in protein binding and function

2014

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Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may "select" protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on-and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or singlemolecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction.

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Conformational selection and induced changes along the catalytic cycle of Escherichia coli dihydrofolate reductase

Cited by 23 publications

References 76 publications

Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Engineered control of enzyme structural dynamics and function

Conformational selection in protein binding and function

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