414 ISSN 1063-7745, Crystallography Reports, 2016, Vol. 61, No. 3, pp. 414–420. © Pleiades Publishing, Inc., 2016. Original Russian Text © L.A. Dadinova, E.V. Rodina, N.N. Vorobyeva, S.A. Kurilova, T.I. Nazarova, E.V. Shtykova, 2016, published in Kristallografiya, 2016, Vol. 61, No. 3, pp. 406–412. Structural Investigations of E. Coli Dihydrolipoamide Dehydrogenase in Solution: Small-Angle X-Ray Scattering and Molecular Docking L. A. Dadinova a,b , E. V. Rodina b , N. N. Vorobyeva b , S. A. Kurilova b , T. I. Nazarova b , and E. V. Shtykova a,b a Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninskii pr. 59, Moscow, 119333 Russia b Moscow State University, Moscow, 119992 Russia e-mail: shtykova@ns.crys.ras.ru Received December 10, 2015 Abstract—Dihydrolipoamide dehydrogenase from Escherichia coli (LpD) is a bacterial enzyme that is involved in the central metabolism and shared in common between the pyruvate dehydrogenase and 2-oxo- glutarate dehydrogenase complexes. In the crystal structure, E. coli LpD is known to exist as a dimer. The present work is focused on analyzing the solution structure of LpD by small-angle X-ray scattering, molecular docking, and analytical ultracentrifugation. It was shown that in solution LpD exists as an equilibrium mix- ture of a dimer and a tetramer. The presence of oligomeric forms is determined by the multifunctionality of LpD in the cell, in particular, the required stoichiometry in the complexes. DOI: 10.1134/S1063774516030093 INTRODUCTION Dihydrolipoamide dehydrogenase (LpD) is a fla- vin-containing protein that catalyzes the oxidation of the SH group of lipoamide in oxidoreductases to form an S–S bond. The flavin cofactor (FAD) of LpD is reduced to FADH 2 , and its reverse conversion requires the presence of NAD + . Dihydrolipoamide dehydroge- nase is involved in three different multienzyme com- plexes that catalyze similar decarboxylation reactions of 2-oxoacids. All of these complexes comprise three enzymes known as Е1, Е2, and Е3, where LpD is the Е3 component and the E2 subunit is used by LpD as the lipoamide-containing protein substrate. The Е1 and Е2 subunits have different structures in different complexes, whereas the Е3 protein is essentially the same in all of the complexes [1–4]. The two main Е3- containing complexes play an essential metabolic role. Thus, they link the utilization of carbohydrate sub- strates to oxidative phosphorylation. The pyruvate dehydrogenase complex (PDC) converts pyruvate (the glycolysis product) to Ас-СоА (the main substrate of the Krebs cycle). The 2-oxoglutarate dehydrogenase complex catalyzes one of the reactions in the Krebs cycle. The pyruvate dehydrogenase complex from Gram-negative bacteria (for example, from E. coli) is composed of 24 E1 subunits and 24 E2 subunits, whereas the multiplicity of E3 remains unknown. According to different estimates, there are 12 [5, 6] or 24 [7–9] E3 subunits; i.e., E3 may consist of six dimers or six tetramers. Meanwhile, all of the Е3 enzymes function as dimers, and their active site con- tains the reactive disulfide bridge, which is directly involved in catalysis [10]. These reactions are essential for aerobic respiration. Dihydrolipoamide dehydroge- nase plays an equally important role in anaerobic organisms, since this enzyme is involved in the syn- thesis of branched-chain keto and amino acids. The E3 enzyme was also found in the absence of multien- zyme complexes, for example, in organisms lacking 2-oxoacid dehydrogenase [11, 12]. These properties attest to the multifunctional character of LpD. The crystal structure of E. coli LpD was determined at 2.5-Å resolution [10], and it was shown that LpD exists as a dimer in the crystalline state (PDB ID: 4JDR). However, the solution structure of this protein was unknown. Since all vitally essential reactions of enzymes occur in a liquid medium, the aim of the present study is to investigate the behavior of LpD in solution, i.e., under near-physiological conditions, by small-angle X-ray scattering (SAXS). The SAXS method is suit- able for the determination of a complex molecular architecture of proteins even in a multicomponent solution [13]. Modern techniques for the interpreta- tion of SAXS data provide information about the qua- ternary structure of biological objects. For example, molecular tectonics can be employed to determine the structures of macromolecular complexes composed of subunits with known or partially known atomic struc- tures [13–16]. However, it should be taken into account that different models may be reconstructed from the same SAXS curve because the recovery of a three-dimensional object from a one-dimensional curve is obviously ambiguous [17]. In order to reduce the ambiguity in the interpretation of the SAXS data, STRUCTURE OF MACROMOLECULAR COMPOUNDS