Articles Communication between the Nucleotide Site and the Main Molecular Hinge of 3-Phosphoglycerate Kinase † Judit Szabo ´, ‡ Andrea Varga, ‡ Bea ´ta Flachner, ‡ Peter V. Konarev, § Dmitri I. Svergun, § Pe ´ter Za ´vodszky, ‡ and Ma ´ria Vas* ,‡ Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary, EMBL Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany, and Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia ReceiVed March 10, 2008; ReVised Manuscript ReceiVed April 25, 2008 ABSTRACT: 3-Phosphoglycerate kinase is a hinge-bending enzyme with substrate-assisted domain closure. However, the closure mechanism has not been described in terms of structural details. Here we present experimental evidence of the participation of individual substrate binding side chains in the operation of the main hinge which is distant from the substrate binding sites. The combined mutational, kinetic, and structural (DSC and SAXS) data for human 3-phosphoglycerate kinase have shown that catalytic residue R38, which also binds the substrate 3-phosphoglycerate, is essential in inducing domain closure. Similarly, residues K219, N336, and E343 which interact with the nucleotide substrates are involved in the process of domain closure. The other catalytic residue, K215, covers a large distance during catalysis but has no direct role in domain closure. The transmission path of the nucleotide effect toward the main hinge of PGK is described for the first time at the level of interactions existing in the tertiary structure. Domain movement is the most spectacular phenomenon of protein flexibility and is essential for the function of most multidomain proteins, including enzymes (cf. reviews 1–3). Domain closure is generally regulated by sophisticated molecular mechanisms, descriptions of which are required to fully understand the structure-function relationship of multidomain proteins. 3-Phosphoglycerate kinase (PGK) 1 is the subject of this investigation and is a typical hinge-bending enzyme with two interacting structural domains. PGK catalyzes the phospho transfer from 1,3-bisphosphoglycerate (1,3-BPG) to MgADP and produces 3-phosphoglycerate (3- PG) and MgATP during glycolysis. In addition, PGK plays an important role in the phosphorylation of L-nucleoside analogues which are drug molecules against cancer and viral infections (4, 5). The PGK domains are approximately equal in size (6–8), and each of them binds one of the two substrates. 3-PG (9) and 1,3-BPG interact with the N-terminal domain, while the nucleotide substrates [MgATP (10) or MgADP (11)] bind to the C-terminal domain (cf. Figure 1A). Two extreme conformations (open and closed) of the structure of PGK (from different sources) are known (12–15). In the open structure, the two bound substrates are distant from each other for the reaction to occur. However, upon closure of the domains, the reactive groups of the substrates move together and become correctly oriented for the catalyzed phospho transfer reaction, as suggested in one of the closed crystal structures (14). By comparing different pairs of open and closed crystal structures, Szila ´gyi et al. proposed that -strand L (located between the two domains) operates as the main hinge of PGK, since the conformation of L determines the relative position of the two domains (15) (Figure 1A). Location of a hinge at L has been supported with molecular modeling using DynDom 2 (2, 16, 17). However, besides L, the molecular modeling also identified another possible hinge at the C-terminus of helix 7 which connects the two domains. † The financial support of Grants OTKA (T 043446, T 046412, NI 61915, and D 048578) from the Hungarian National Research Fund, Grant GVOP-3.2.1.2004-04 0195/3.0, and the traveling grants for B.F., J.Sz., and A.V. provided by the European Community-Research Infrastructure Action, under FP6 “Structuring the European Research Area Programme” Contract RII3/CT/2004/5060008, is gratefully acknowledged. D.I.S. and P.V.K. acknowledge support from the EU Design Study “SAXIER”, Contract 011934. * To whom correspondence should be addressed: Institute of Enzymology, BRC, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary. Telephone: 36 1 279 3152. Fax: 36 1 466 5465. E-mail: vas@enzim.hu. ‡ Hungarian Academy of Sciences. § EMBL Outstation, c/o DESY, and Russian Academy of Sciences. 1 Abbreviations: AMP-PNP, ,γ-imidoadenosine 5′-triphosphate; ANS, 1-anilinonaphthalene-8-sulfonic acid; 1,3-BPG, 1,3-bisphospho- glycerate; CD, circular dichroism; DSC, differential scanning calorim- etry; DTT, dithiothreitol; GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); G6PDH, glucose-6-phosphate 1-dehy- drogenase (EC 1.1.1.49); HK, hexokinase (EC 2.7.1.1); 3-PG, 3-phos- phoglycerate; PGK, 3-phospho-D-glycerate kinase or ATP, 3-phospho- D-glycerate 1-phosphotransferase (EC 2.7.2.3); hPGK, human PGK; SAXS, small-angle X-ray scattering; Tb, Trypanosoma brucei; Tm, Thermotoga maritima; Bs, Bacillus stearothermophilus. 2 See http://www.cmp.uea.ac.uk/dyndom. Biochemistry 2008, 47, 6735–6744 6735 10.1021/bi800411w CCC: $40.75  2008 American Chemical Society Published on Web 06/10/2008