Dissecting the molecular determinants of ligand-binding-induced macromolecular switching using thermodynamic cycles

Smith, Martin T. J.; MacKenzie, Duncan; Meiering, Elizabeth M.

doi:10.1093/protein/gzq099

Cited by 6 publications

(9 citation statements)

References 24 publications

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“…We then analyzed the contributions of individual residues to switching, using a combination of experimental measurements and atomistic simulations for mutant proteins. In previous experiments, we found that the switch in hisactophilin is broken in the triple myristoyl binding pocket mutant, F6L/I85L/I93L, such that the myristoyl group remains in the sequestered state and does not switch to the accessible state with decreasing pH (25) (Fig. 3C).…”

Section: Resultsmentioning

confidence: 70%

“…Based on previous analyses of the pH dependence of stability and NMR data, switching from the sequestered state to the accessible state is caused by the binding of approximately 1.5 protons by a small number of histidine residues localized on one side of the protein (6,25). Also, the protonation of the histidines is coupled to the state of nearby hydrophobic residues including F6, I85 and I93, which sensitively communicate pH changes to the myristoyl binding pocket as evidenced by switching being abolished in the F6L/I85L/I93L (25) and I85L (Fig. 3C) mutants.…”

Section: Resultsmentioning

confidence: 99%

“…1B). Variants include a triple myristoyl binding pocket mutant (F6L/I85L/I93L) in which pHdependent switching is abolished (25), and single mutations that alter stereochemistry (I85L) or truncate side chains inside (V36A and L76A) or outside (I118A) the binding pocket. The results provide unique insight into the role of nonnative interactions in folding as well as the atomistic mechanism of myristoyl switching.…”

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confidence: 99%

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Nonnative interactions regulate folding and switching of myristoylated protein

Shental-Bechor

Smith

MacKenzie

et al. 2012

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

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We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin-and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.coarse-grained simulation | funnel landscape | β-trefoil P rotein folding is governed by various physicochemical forces that bias the native state, which for many proteins is also the functional state, over the many alternative nonnative states. The network of native interactions has been found in many cases to be sufficient to capture the folding mechanism and kinetics of proteins (1, 2). The discrimination between native and nonnative interactions is the foundation of the principle of minimal frustration (3) and explains the power of native topology-based models in studying folding biophysics (4). The dominant role of native interactions is manifested by the funnel-shaped energy landscape for folding that suggests folding is robust and an efficient process. The information stored in the native topology may, however, be tuned by various factors such as confining the protein in a small space, crowding agents, or conjugating the protein to other biomolecules [e.g., oligosaccharides (5) or fatty acyl chains such as myristoyl (6)]. In addition to manipulating folding characteristics by modifications or environmental conditions, nonnative interactions, which are by definition in conflict with the native state, may decorate the folding funnel (7, 8) by increasing energetic frustration (9) between interactions and therefore landscape roughness. The degree of roughness, which affects the trapping of the protein in nonnative states, depends on the particular sequence of the pr...

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Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Nonnative interactions regulate folding and switching of myristoylated protein

Shental-Bechor

Smith

MacKenzie

et al. 2012

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

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“…The use of such conditions frequently irreversibly damages the protein reagent and/or the target molecule of interest or is simply not compatible with the reaction/assays of interest. Consequently, the development of protein molecular switches,2, 3 which are proteins whose function (e.g., binding) may be modulated (or linked) through an environmental trigger (e.g., pH or ligand) or conformational change, are desired in many applications.…”

Section: Introductionmentioning

confidence: 99%

A combinatorial histidine scanning library approach to engineer highly pH‐dependent protein switches

et al. 2011

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There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK a change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine ''hot-spots,'' which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.

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“…The study of the pH-dependent catalysis helps in identifying the favorable electrostatic interactions which build up the environment for facilitating the catalytic reaction. Knowledge of the residues that govern various pH-dependent functions could allow us to re-engineer these functions 21 which have several industrial and pharmaceutical applications [22][23][24][25] .…”

Section: Introductionmentioning

confidence: 99%

Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

Roitberg¹,

Gupta²

2019

Preprint

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<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

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Dissecting the molecular determinants of ligand-binding-induced macromolecular switching using thermodynamic cycles

Cited by 6 publications

References 24 publications

Nonnative interactions regulate folding and switching of myristoylated protein

Nonnative interactions regulate folding and switching of myristoylated protein

A combinatorial histidine scanning library approach to engineer highly pH‐dependent protein switches

Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

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