Principles of Protein Stability and Their Application in Computational Design

Goldenzweig, Adi; Fleishman, Sarel J.

doi:10.1146/annurev-biochem-062917-012102

Cited by 220 publications

(223 citation statements)

References 146 publications

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“…Three of the mutations used by NDM variants likely achieve stabilization by either optimizing hydrophobic side chain packing (M154L), as shown using X-ray crystallography (5), or by burying additional hydrophobic surface into the core of the protein (A233V, V88L). Other proteins that have evolved increased thermostability have used similar strategies (19). The contribution of these three particular NDM mutations appear to be additive, since the individual contributions of M154L (Tm, NDM-4 -Tm, NDM-1 = 3.9 ± 0.3 °C), A233V (Tm, NDM-6 -Tm, NDM-1 = 1.8 ± 0.3 °C) and V88L (Tm, NDM-5 -Tm, NDM-4 = 3.5 ± 0.2 °C) can account for the thermostability of the triple mutant (NDM-16 (Tm, exptl = 9.5 ± 0.3, Tm, calc'd = 9.2 ± 0.8).…”

Section: Discussionmentioning

confidence: 99%

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Cheng

Thomas²,

et al. 2018

Journal of Biological Chemistry

107

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Infections by carbapenem-resistant Enterobacteriaceae (CRE) are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically-used β-lactamase inhibitors to counter the activity of metallo-β-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-β-lactamase (NDM), which requires a dinuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing comparing melting temperature and refolding efficiency and also by spectroscopy (UV-Vis, 1 H-NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining of minimum inhibitory concentrations of various antibiotics, pre-steady state and steadystate kinetics, inhibitor binding, and zincdependence of resistance and activity. We observed only minor differences among the fullloaded dizinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants http://www.jbc.org/ Downloaded from 2 exhibited improved thermostability (up to ~10 °C increased Tm) and improved zinc affinity (up to ~10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as monozinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/KM = 5 × 10 6 M -1 s -1 ). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of β-lactam antibiotics and zinc deprivation.Carbapenem-resistant Enterobacteriaceae (CRE) continue to be classified as an "urgent threat," the highest hazard level assigned by the Centers for Disease Control and Prevention(1). The five carbapenemases currently of primary public concern include Klebsiella pneumonia carbapanemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integrin encoded metallo-β-lactamase (VIM), imipenemase (IMP), and oxacillinase-48-like carbapenemase (OXA-48)(2). Three of these carbapenemases (NDM, VIM, and IMP) are metal-dependent β-lactamases that are not susceptible to any of the β-lactamase inhibitors incorporated into combination drugs used in the clinic. Of these three β-lactamases, NDM is the most widespread in U.S. patients, with infections bearing a blaNDM gene reported in 34 / 50 states (as of December 2017)(3).The genes encoding NDM continue to evolve, with discovery of more than 20 variants (NDM-1 through NDM-21 at the time of writing, 16 at the start of this project). Most of these mutations occur at sites distant from the active site, and the functions they confer are not immediately obvious. A comparison of NDM-1 through NDM-8 showed only minor differences in kcat/KM values (≤ 5-fold) for a panel of diverse β-lactam drugs(4). However, a considerable increase in thermostability was noted for many of the variants, suggesting the functional impact of NDM...

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Section: Discussionmentioning

confidence: 99%

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Cheng

Thomas²,

et al. 2018

Journal of Biological Chemistry

107

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“…The introduction of seven simultaneous mutations could severely destabilize the protein . We found, however, that BSG des7 exhibited a high apparent melting temperature (50°C compared to 55°C for BSG), demonstrating that the design procedure maintained protein stability (Figure C).…”

Section: Resultsmentioning

confidence: 85%

Design of a basigin‐mimicking inhibitor targeting the malaria invasion protein RH5

et al. 2019

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Many human pathogens use host cell‐surface receptors to attach and invade cells. Often, the host‐pathogen interaction affinity is low, presenting opportunities to block invasion using a soluble, high‐affinity mimic of the host protein. The Plasmodium falciparum reticulocyte‐binding protein homolog 5 (RH5) provides an exciting candidate for mimicry: it is highly conserved and its moderate affinity binding to the human receptor basigin (KD ≥1 μM) is an essential step in erythrocyte invasion by this malaria parasite. We used deep mutational scanning of a soluble fragment of human basigin to systematically characterize point mutations that enhance basigin affinity for RH5 and then used Rosetta to design a variant within the sequence space of affinity‐enhancing mutations. The resulting seven‐mutation design exhibited 1900‐fold higher affinity (KD approximately 1 nM) for RH5 with a very slow binding off rate (0.23 h−1) and reduced the effective Plasmodium growth‐inhibitory concentration by at least 10‐fold compared to human basigin. The design provides a favorable starting point for engineering on‐rate improvements that are likely to be essential to reach therapeutically effective growth inhibition.

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“…Note that as observed in studies of mutational effects on stability in soluble proteins, the correlation coefficient between computed and observed values was low (Pearson r 2 =0.21 and 0.02 for ref2015_memb and RosettaMembrane, respectively) [51][52][53][54] . Such low correlation coefficients provide an impetus for improving the energy function; however, as we previously demonstrated, discriminating stabilizing from destabilizing mutations is sufficient to enable the design of accurate, stable, and functionally efficient proteins [54][55][56][57][58][59] . We next tested sequence-recovery rates using combinatorial sequence optimisation based on ref2015, ref2015_memb, and RosettaMembrane in a benchmark of 20 non-redundant structures (<80% sequence identity) ranging in size from 124-765 amino acids 60 .…”

Section: Ab Initio Structure Prediction In Membrane Proteinsmentioning

confidence: 93%

A lipophilicity-based energy function for membrane-protein modelling and design

Weinstein

Elazar

Fleishman

2019

Preprint

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Membrane-protein design is an exciting and increasingly successful research area which has led to landmarks including the design of stable and accurate membrane-integral proteins based on coiled-coil motifs. Design of topologically more complex proteins, such as most receptors, channels, and transporters, however, demands an energy function that balances contributions from intra-protein contacts and protein-membrane interactions. Recent advances in water-soluble all-atom energy functions have increased the accuracy in structure-prediction benchmarks. The plasma membrane, however, imposes different physical constraints on protein solvation. To understand these constraints, we recently developed a high-throughput experimental screen, called dsTβL, and inferred apparent insertion energies for each amino acid at dozens of positions across the bacterial plasma membrane. Here, we express these profiles as lipophilicity energy terms in Rosetta and demonstrate that the new energy function outperforms previous ones in modelling and design benchmarks. Rosetta ab initio simulations starting from an extended chain recapitulate two-thirds of the experimentally determined structures of membrane-spanning homo-oligomers with <2.5Å root-mean-square deviation within the top-predicted five models. Furthermore, in two sequence-design benchmarks, the energy function improves discrimination of stabilizing point mutations and recapitulates natural membrane-protein sequences of known structure, thereby recommending this new energy function for membrane-protein modelling and design.

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Principles of Protein Stability and Their Application in Computational Design

Cited by 220 publications

References 146 publications

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Design of a basigin‐mimicking inhibitor targeting the malaria invasion protein RH5

A lipophilicity-based energy function for membrane-protein modelling and design

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