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Biochimica et Biophysica Acta 1595 (2002) 382^386 www.bba-direct.com Review Compressibility gives new insight into protein dynamics and enzyme function Kunihiko Gekko * Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan Received 16 November 2001; accepted 19 November 2001 Abstract The adiabatic compressibility of enzyme is largely influenced by binding of coenzyme and substrate, due to the changes in atomic packing. Amino acid substitution also induces large changes in compressibility parallel to enzyme activity. These results demonstrate that a small alteration of local structure by ligand binding and mutation is dramatically magnified in the flexibility of protein molecule to affect the function. Compressibility gives new insight into protein dynamics and enzyme function from the aspect of atomic packing or cavity which cannot be obtained by other techniques. ß 2002 Elsevier Science B.V. All rights reserved. Keywords: Adiabatic compressibility; Amino acid substitution; Cavity; Enzyme function; Ligand binding; Protein dynamics 1. Introduction Compressibility is not only a basic physical quantity to analyze the pressure e¡ects on protein structure, but also it gives new insight into protein dynamics which contributes to biological function of protein molecules. According to the X-ray crystal structure, globular proteins have a precisely de¢ned compact structure and all atoms in the molecule seem to be closely packed as well as the crystalline amino acids. However, there are many packing defects or cavities in the interior of a protein molecule, which cause internal motions or £exibility response to thermal or mechanical forces. Such cavities are easily compressed by pressure, so compressibility is * Fax: +81-824-247387. E-mail address: [email protected] (K. Gekko). an important measure of protein £exibility or volume £uctuation. According to statistical thermodynamics, the volume £uctuation NVrms of a protein with volume Vp is related to its isothermal compressibility LT [1]: N V rms ¼ ðkTV p L T Þ1=2 ð1Þ where k is the Boltzmann constant and T the absolute temperature. Measurement of compressibility is possible, in principle, only for proteins in solution. Although volume £uctuation is directly linked to isothermal compressibility, most compressibility studies of proteins have been of adiabatic compressibility because of the accuracy and technical convenience [2^5]. The partial speci¢c adiabatic compressibility Ls ‡ is de¢ned as the change in the partial speci¢c volume v‡ with pressure. Since the constitutive atomic volume of a protein may be regarded as incompressible, the experimen- 0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 3 5 8 - 2 BBAPRO 36578 23-4-02 K. Gekko / Biochimica et Biophysica Acta 1595 (2002) 382^386 tally determined Ls ‡ can be mainly attributed to its cavity vcav and to changes in hydration vvsol [3], L s ¼ 3ð1=v ÞðD v =D PÞ ¼ 3ð1=v Þ½D vcav =D P þ D v vsol =D P ð2Þ The cavity contributes positively and hydration negatively to v‡ and Ls ‡, then Ls ‡ can be positive or negative depending on the magnitude of both terms. Most globular proteins have a positive Ls ‡ value of 0^14 Mbar31 at 25‡C, indicating the large contribution of cavity overcoming the hydration e¡ect [3]. The volume £uctuation is in the range from 30 to 200 cm3 mol31 , which correspond to only 0.3% of whole protein volume. But if concentrated in one area, it would produce su⁄cient cavities or channels in the protein molecule to allow penetration of solvent or probe molecules. Recent studies on the e¡ects of ligand binding and amino acid substitution on Ls ‡ demonstrate that a small alteration of local structure is dramatically magni¢ed in the overall protein dynamics to a¡ect the enzyme function [6^9]. 2. E¡ects of ligand binding The enzyme^ligand complex formation generally proceeds in three states: formation of net bonds between enzyme and ligand, changes in hydration of the interacting species, and conformational changes of the enzyme. The solute-solute interaction in water should accompany dehydration of each solute molecule, resulting in the increase in v‡ and Ls ‡. However, the observed changes in both parameters by ligand binding are not necessarily positive. A typical example is lysozyme^inhibitor complex formation. The v‡ and Ls ‡ values of lysozyme decrease by addition of N-acetyl-D-glucosamine oligomers, in the order of monomer s dimer s trimer [6]. Assuming that the decrease in Ls ‡ is ascribed to the protein moiety only, the volume £uctuation is estimated to decrease by about 3%, 7%, and 14% on binding the monomer, dimer and trimer, respectively. The X-ray crystallographic study shows that the inhibitors are bound in the cleft of the active site of lysozyme. The e¡ects of this ‘induced-¢t’ type interaction would propagate throughout the entire protein molecule to enhance the internal atomic packing, resulting in the de- 383 pressed £uctuation of the protein molecule. The hydrogen-exchange rate of lysozyme is also known to decrease in the same order of these oligomers [10]. It is noteworthy that the volume £uctuation changes comparably to the kinetically determined £uctuation. More pronounced changes in Ls ‡ and v‡ are found on ligand binding of Escherichia coli dihydrofolate reductase (DHFR). DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) with the aid of coenzyme NADPH in the kinetic reactions cycling through ¢ve intermediates, DHFRWNADPH, DHFRWNADPHWDHF, DHFRW NADPþ WTHF, DHFRWTHF, and DHFRW NADPHW THF [11]. This enzymatic function is strongly repressed with an inhibitor, methotrexate (MTX). The X-ray structure of a DHFRWNADPHWMTX ternary complex is shown in Fig. 1 [12]. The Ls ‡ of these kinetic intermediates is shown as a function of the reaction coordinate in Fig. 2 [7], where the transient state DHFRW NADPHWDHF is substituted by a complex DHFRWNADPHWMTX since the X-ray structures of both complexes are quite similar. Evidently, the £exibility of DHFR changes alternatively by binding or releasing the coenzyme and substrate: the transient state DHFRWNADPHWDHF is expected to be most £exible and the state DHFRWNADPþ W THF most rigid in the intermediates. As illustrated in Fig. 3, the number, size and distribution of cavities also change on ligand binding, parallel to the changes in partial speci¢c volume (vv‡) and compressibility (vKs = vv‡Ls ‡). On binding of these ligands, the total cavity volume is largely in£uenced ( þ 40%) but the solvent accessible surface area only slightly decreases (at most 6%), indicating that the cavity plays dominant role in ligand-induced compressibility change overcoming hydration e¡ects. Recent high pressure NMR study of folate-bound DHFR reveals that major conformation changes by pressure occur in the NADPH binding site and the £exible loops [13]. 3. E¡ects of amino acid substitution How does the single amino acid substitution a¡ect the £exibility of overall protein structure? A key to answer this problem is found in the mutation e¡ects on Ls ‡ of two E. coli enzymes, aspartate aminotrans- BBAPRO 36578 23-4-02 384 K. Gekko / Biochimica et Biophysica Acta 1595 (2002) 382^386 Fig. 2. The Ls ‡ level of the kinetic intermediates of DHFR as a function of the reaction pathway [7]. Fig. 1. The structure of a DHFR^MTX^NADPH ternary complex taken from Sawaya and Kraut [12]. Positions Gly67, Gly121, Ala145 (mutation sites) and Asp27 (catalytic site) are indicated by arrows. ferase (AspAT) and DHFR. AspAT has two identical 45 kDa subunits and the catalytic site is formed between the large and small domains in each subunit. Six mutant enzymes at Val39, which is located at the gate of the substrate binding site, have evidently different Ls ‡ values [8]. The observed positive linear correlation between Ls ‡ and van der Waals volume of the introduced amino acids suggests that the bulkiness of the side chain at position 39 makes the protein structure more £exible due to the overcrowding e¡ect in atomic packing. The catalytic e⁄ciency of AspAt is also modi¢ed by these mutations, where major changes occur on Km but not on kcat . Interestingly, there is a linear correlation between log (kcat / Km ) and Ls ‡, showing that the amino acid substitution at Val39, which increases £exibility of overall structure, makes the enzyme more active. The enzyme activity of DHFR is known to be in£uenced by mutations at Gly67, Gly121, and Ala145, which are located at the center of three different £exible loops (see Fig. 1), where Km is only slightly in£uenced but kcat remarkably changes [14^ 16]. This is very interesting because it is unlikely that the mutation sites directly participate in the enzyme reaction: K-carbons of Gly67, Gly121 and Ala145 î are separated respectively by 29.3, 19.0 and 14.4 A from the catalytic site Asp27, and at shortest by 8.5, î from the NADPH molecule. Fur10.6 and 29.2 A thermore, double mutants at Gly67 and Gly121, î, whose K-carbons are mutually separated by 27.7 A show non-additive e¡ects on the function [17]. These ¢ndings suggest that these mutations would alter the Fig. 3. The changes of number (n), position and total volume (vVcav ) of cavities in some kinetic intermediates of DHFR [7]. vKs = vv‡Ls ‡: cm3 g31 Mbar31 . BBAPRO 36578 23-4-02 K. Gekko / Biochimica et Biophysica Acta 1595 (2002) 382^386 385 local structure due to ligand binding and amino acid substitution is dramatically magni¢ed in the overall protein dynamics, possibly via ‘stepwise atomic reconstruction or long-range cooperative e¡ect’, leading to modi¢cation of catalytic activity of enzymes. Compressibility study in combination with X-ray crystallography and high-pressure NMR would be fruitful for elucidating the structure^£exibility^function relationship of enzymes. References Fig. 4. Plots of Km and log (kcat /Km ) against Ls ‡ of DHFR mutants [9]. £exibility of the entire protein molecule. In fact, these mutations induce large changes in v‡ (0.710^ 0.733 cm3 g31 ) and Ls ‡ (31.8^5.5 Mbar31 ) from the corresponding values of wild-type enzyme (v‡ = 0.723 cm3 g31 , Ls ‡ = 1.7 Mbar31 ) [9]. As shown in Fig. 4, there is a de¢nite correlation between Ls ‡ and Km or log (kcat /Km ), indicating that the structural £exibility positively contributes to the enzyme function, as is the case of AspAT, through an enhanced catalytic reaction rate and in part due to increased a⁄nity for the substrate. It is important that the £exibility-mediated modi¢cation of enzyme activity can be derived by the mutation at the loop regions far from the catalytic site because this gives a new strategy to design more functional proteins. Our recent X-ray analyses reveal that the B-factors of main chain atoms and the cavities at positions far from the mutation sites are modi¢ed without any large change in solvent accessible surface area and the mutants with the large Ls ‡ values have large amount of cavity (K. Katayanagi et al., to be published). These results demonstrate that the e¡ects of mutation extend to a long range and the mutation-induced Ls ‡ change is dominantly ascribed to modi¢cation of internal cavities. As shown here, compressibility gives new insight into protein dynamics and enzyme function from the aspect of atomic packing or cavity which cannot be obtained by other techniques. A small alteration of [1] A. Cooper, Thermodynamic £uctuations in protein molecules, Proc. Natl. Acad. Sci. USA 73 (1976) 2740^2741. [2] K. Gekko, H. Noguchi, Compressibility of globular proteins in water at 25‡C, J. Phys. Chem. 83 (1979) 2706^2714. [3] K. Gekko, Y. Hasegawa, Compressibility-structure relationship of globular proteins, Biochemistry 25 (1986) 6563^ 6571. [4] T.V. Chalikian, V.S. Gindikin, K.J. Breslauer, Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH, J. Mol. Biol. 250 (1995) 291^306. [5] D.P. Kharakoz, Partial volumes and compressibilities of extended polypeptide chains in aqueous solution: additivity scheme and implication of protein unfolding at normal and high pressure, Biochemistry 36 (1997) 10276^10285. [6] K. Gekko, K. Yamagami, Compressibility and volume changes of lysozyme due to inhibitor binding, Chem. Lett. (1998) 839-840. [7] T. Kamiyama, K. Gekko, E¡ect of ligand binding on the £exibility of dihydrofolate reductase as revealed compressibility, Biochim. Biophys. Acta 1478 (2000) 257^266. [8] K. Gekko, Y. Tamura, E. Ohmae, H. Hayashi, H. Kagamiyama, H. 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Akasaka, High pressure NMR reveals active-site hinge BBAPRO 36578 23-4-02 386 K. Gekko / Biochimica et Biophysica Acta 1595 (2002) 382^386 motion of folate-bound Escherichia coli dihydrofolate reductase, Biochemistry 39 (2000) 12789^12795. [14] K. Gekko, Y. Kunori, H. Takeuchi, S. Ichihara, M. Kodama, Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a £exible loop in the stability and function, J. Biochem. 116 (1994) 34^41. [15] E. Ohmae, K. Iruyama, K. Ichihara, K. Gekko, E¡ects of point mutations at the £exible loop of Escherichia coli dihydrofolate reductase on its stability and function, J. Biochem. 119 (1996) 703^710. [16] E. Ohmae, K. Ishimura, M. Iwakura, K. Gekko, E¡ects of point mutations at the £exible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function, J. Biochem. 123 (1998) 839^846. [17] E. Ohmae, K. Iriyama, S. Ichihara, K. Gekko, Nonadditive e¡ects of double mutations at the £exible loops, glycine-67 and glycine-121, of Escherichia coli dihydrofolate reductase on its stability and function, J. Biochem. 123 (1998) 33^41. BBAPRO 36578 23-4-02