Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system

Chu, V.; Freitag, Stefanie; Trong, I. Le; Stenkamp, Ronald E.; Stayton, Patrick S.

doi:10.1002/pro.5560070403

Cited by 76 publications

(80 citation statements)

References 45 publications

(39 reference statements)

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“…This finding is consistent with a destabilization of the water ring solvating the active site due to the loss of the hydrogen binding groups maintaining it (8). Circular deletion of the mobile loop formed by residues 47-51 that hydrophobically enclose the ligand from above, most notably by Val-47, loses 8 kcal/mol lower than the unmutated protein; this is much more than continuum methods predict but in agreement with our estimate (9). Mutating Trp-79 to Phe was found to enthalpically stabilize biotin binding by 1.5 kcal/mol but entropically destabilize it by 2.4 kcal/mol (10).…”

Section: Resultssupporting

confidence: 90%

Motifs for molecular recognition exploiting hydrophobic enclosure in protein–ligand binding

Young

Abel

Kim

et al. 2007

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

645

839

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The thermodynamic properties and phase behavior of water in confined regions can vary significantly from that observed in the bulk. This is particularly true for systems in which the confinement is on the molecular-length scale. In this study, we use molecular dynamics simulations and a powerful solvent analysis technique based on inhomogenous solvation theory to investigate the properties of water molecules that solvate the confined regions of protein active sites. Our simulations and analysis indicate that the solvation of protein active sites that are characterized by hydrophobic enclosure and correlated hydrogen bonds induce atypical entropic and enthalpic penalties of hydration. These penalties apparently stabilize the protein-ligand complex with respect to the independently solvated ligand and protein, which leads to enhanced binding affinities. Our analysis elucidates several challenging cases, including the super affinity of the streptavidin-biotin system.binding motifs ͉ hydrophobic effect ͉ streptavidin ͉ dewetting T he hydrophobic interaction is considered to be an important driving force in molecular recognition, yet our understanding of hydrophobicity in enclosed regions, such as those found in protein binding sites, remains incomplete. For example, the binding affinity of biotin to streptavidin is orders of magnitude larger than expected on the basis of most current theoretical models. The inability to predict such ''super affinities'' and the absence of a molecular understanding of hydrophobic enclosure effects stands as an obstacle to rational design of potent pharmacologically active compounds. A better understanding of the nature of such enclosures is essential to further progress in the area. We show how superaffinity can arise from active sites that have two important molecular recognition motifs: hydrophobic enclosure and correlated hydrogen bonds. Using molecular dynamics, we show that these motifs can induce atypical entropic and enthalpic penalties for hydration of the apostructures of proteins that stabilize the bound state with respect to the hydrated state and, hence, lead to super affinity.It is widely believed that hydrophobic interactions constitute the principal thermodynamic driving force for the binding of small molecule ligands to their cognate protein receptors. A substantial number of empirical scoring functions aimed at computing protein-ligand binding affinities have been developed; invariably, the largest contribution in such expressions represents a measure of hydrophobic contact between the protein and ligand (1). Underlying these contributions is the idea that replacement of water molecules in the protein cavity by a ligand that is complementary to the protein groups lining the cavity (making hydrogen bonds where appropriate, and hydrophobic contacts otherwise) leads to a gain in binding affinity by releasing water molecules from a suboptimal environment into solution. Standard scoring functions aimed at describing this effect are based on pairwise atom-atom terms or bur...

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Section: Resultssupporting

confidence: 90%

Motifs for molecular recognition exploiting hydrophobic enclosure in protein–ligand binding

Young

Abel

Kim

et al. 2007

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

645

839

View full text Add to dashboard Cite

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“…Furthermore, the water channel is lined by residues of the ordered loop 2, which can aid the channel to act as a proton relay system. This proposal is in accordance with the generally accepted view that loop closures separate the bulk water from the reaction region (24,29,30). In such models, water is either squeezed out of the active site, or a limited number of selected water molecules are retained for catalysis.…”

Section: Discussionsupporting

confidence: 86%

A Flexible Loop as a Functional Element in the Catalytic Mechanism of Nucleoside Hydrolase from Trypanosoma vivax

Vandemeulebroucke¹,

Vos²,

Holsbeke

et al. 2008

Journal of Biological Chemistry

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The nucleoside hydrolase of Trypanosoma vivax hydrolyzes the N-glycosidic bond of purine nucleosides. Structural and kinetic studies on this enzyme have suggested a catalytic role for a flexible loop in the vicinity of the active sites. Here we present the analysis of the role of this flexible loop via the combination of a proline scan of the loop, loop deletion mutagenesis, steady state and pre-steady state analysis, and x-ray crystallography. Our analysis reveals that this loop has an important role in leaving group activation and product release. The catalytic role involves the entire loop and could only be perturbed by deletion of the entire loop and not by single site mutagenesis. We present evidence that the loop closes over the active site during catalysis, thereby ordering a water channel that is involved in leaving group activation. Once chemistry has taken place, the loop dynamics determine the rate of product release.The importance of conformational changes in enzyme catalysis has long been appreciated. These changes are thought to fulfill a number of roles in catalysis: enhanced binding of substrate, correct orientation of catalytic groups, removal of water from the active site, and trapping of intermediates. Loop motion is classified as an enzyme conformational change, where flexible surface loops move to different conformations (1).Flexible loops are also a common feature of the family of nucleoside hydrolases (EC 3.2.2.1) (2). All structures of the nucleoside hydrolases determined until now show two flexible loops in the vicinity of the active sites, which undergo substantial conformational changes upon association of substrates and inhibitors (2). Nucleoside hydrolases (NHs) 4 catalyze the hydrolysis of the N-glycosidic bond of nucleosides, forming ribose and the corresponding base ( Fig. 1) (2). It has been shown that the NH-catalyzed hydrolysis proceeds via a highly dissociative S N 2-type mechanism with an oxocarbenium ion-like transition state (3, 4). A general catalytic mechanism based on three strategies has been proposed: activation of the nucleophilic water molecule, steric and electrostatic stabilization of the oxocarbenium ion, and leaving group (LG) activation by protonation or hydrogen bonding (2).The purine-specific nucleoside hydrolase of Trypanosoma vivax (TvNH) has been very well characterized kinetically and structurally. This allowed us to put forward a detailed catalytic mechanism to accomplish these strategies. An aspartate (Asp 10 ) in the ribose binding pocket acts as a general base to activate the nucleophile water (5). The oxocarbenium ion is stabilized through interactions between the enzyme and all of the hydroxyl groups (6). The third catalytic strategy, LG activation, was thought to be accomplished by a Brönsted acid, which protonates nitrogen-7 (N-7) of the purine base (2, 7-9). However, in TvNH, no obvious proton donor was found. Here, the LG of the substrate is stacked between the two indole side chains of Trp 83 and Trp 260 ( Fig. 2A). A novel catalytic mechanism...

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“…Removal of the 3-4 loop in streptavidin results in a reduction of the K a by 6 orders of magnitude from wild type, nearly half of the binding energy. 16,36 Molecular dynamics, and separate electrostatic and van der Waals free-energy calculations of the biotin-streptavidin complex suggested that the largest contribution to the extremely negative free energy of binding is the non-polar van der Waals contribution of the tryptophan residues Trp 79, Trp 92, Trp 108, and Trp 120 rather than electrostatic forces. 24,25 The simulations also predict a large electrostatic contribution inside the protein, supported by sitedirected mutagenesis experiments studies on streptavidin, which show that mutation of a residue directly hydrogen-bonded to the ureido moiety of biotin results in a substantial decrease in binding energy.…”

Section: Introductionmentioning

confidence: 99%

The Origins of Femtomolar Protein−Ligand Binding: Hydrogen-Bond Cooperativity and Desolvation Energetics in the Biotin−(Strept)Avidin Binding Site

2007

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The unusually strong reversible binding of biotin by avidin and streptavidin has been investigated by density functional and MP2 ab initio quantum mechanical methods. The solvation of biotin by water has also been studied through QM/MM/MC calculations. The ureido moiety of biotin in the bound state hydrogen bonds to five residues, three to the carbonyl oxygen and one for each -NH group. These five hydrogen bonds act cooperatively, leading to stabilization that is larger than the sum of individual hydrogen-bonding energies. The charged aspartate is the key residue that provides the driving force for cooperativity in the hydrogen-bonding network for both avidin and streptavidin by greatly polarizing the urea of biotin. If the residue is removed, the network is disrupted, and the attenuation of the energetic contributions from the neighboring residues results in significant reduction of cooperative interactions. Aspartate is directly hydrogen-bonded with biotin in streptavidin and is one residue removed in avidin. The hydrogen-bonding groups in streptavidin are computed to give larger cooperative hydrogen-bonding effects than avidin. However, the net gain in electrostatic binding energy is predicted to favor the avidin-bicyclic urea complex due to the relatively large penalty for desolvation of the streptavidin binding site (specifically expulsion of bound water molecules). QM/MM/MC calculations involving biotin and the ureido moiety in aqueous solution, featuring PDDG/PM3, show that water interactions with the bicyclic urea are much weaker than (strept)avidin interactions due to relatively low polarization of the urea group in water.

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Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system

Cited by 76 publications

References 45 publications

Motifs for molecular recognition exploiting hydrophobic enclosure in protein–ligand binding

Motifs for molecular recognition exploiting hydrophobic enclosure in protein–ligand binding

A Flexible Loop as a Functional Element in the Catalytic Mechanism of Nucleoside Hydrolase from Trypanosoma vivax

The Origins of Femtomolar Protein−Ligand Binding: Hydrogen-Bond Cooperativity and Desolvation Energetics in the Biotin−(Strept)Avidin Binding Site

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