Large conformational changes in the Escherichia coli tryptophan synthase b2 subunit upon pyridoxal 5¢-phosphate binding Kazuya Nishio1, Kyoko Ogasahara1, Yukio Morimoto2,3, Tomitake Tsukihara1,4, Soo Jae Lee5 and Katsuhide Yutani3 Institute for Protein Research, Osaka University, Japan Research Reactor Institute, Kyoto University, Japan RIKEN SPring-8 Center, Harima Institute, Japan Department of Life Science, University of Hyogo, Japan College of Pharmacy, Chungbuk National University, Korea Keywords apo- and holo-forms; conformational change; PLP-binding; tryptophan synthase b2 subunit; X-ray crystal structure Correspondence Katsuhide Yutani, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan Fax: 81-791-58-2917 Tel: 81-791-58-2937 E-mail: yutani@spring8.or.jp Soo Jae Lee, College of Pharmacy, Chungbuk National University, Sungbong-ro 410, Cheongju, Chungbuk, Korea Fax: 82-43-268-2732 Tel: 82-43-261-2816 E-mail: sjlee@chungbuk.ac.kr Database Structural data are available from the Protein Data Bank under the accession codes for the holo- (2DH5) and apo- (2DH6) forms (Received 16 October 2009, revised 24 February 2010, accepted March 2010) doi:10.1111/j.1742-4658.2010.07631.x To understand the basis for the lower activity of the tryptophan synthase b2 subunit in comparison to the a2b2 complex, we determined the crystal struc˚ tures of apo-b2 and holo-b2 from Escherichia coli at 3.0 and 2.9 A resolutions, respectively To our knowledge, this is the first report of both b2 subunit structures with and without pyridoxal-5¢-phosphate The apo-type molecule retained a dimeric form in solution, as in the case of the holo-b2 subunit The subunit structures of both the apo-b2 and the holo-b2 forms consisted of two domains, namely the N domain and the C domain Although there were significant structural differences between the apo- and holo-structures, they could be easily superimposed with a 22° rigid body rotation of the C domain The pyridoxal-5¢-phosphate-bound holo-form had multiple interactions between the two domains and a long loop (residues 260–310), which were missing in the apo-form Comparison of the structures of holo-Ecb2 and Stb2 in the a2b2 complex from Salmonella typhimurium (Sta2b2) identified the cause of the lower enzymatic activity of holo-Ecb2 in comparison with Sta2b2 The substrate (indole) gate residues, Tyr279 and Phe280, block entry of the substrate into the b2 subunit, although the indole can directly access the active site as a result of a wider cleft between the N and C domains in the holo-Ecb2 subunit In addition, the structure around bAsp305 of the holo-Ecb2 subunit was similar to the open state of Sta2b2 with low activity, resulting in lower activity of holo-Ecb2 Structured digital abstract l MINT-7712009: Ecb2 (uniprotkb:P0A879) and Ecb2 (uniprotkb:P0A879) bind (MI:0407) by x-ray crystallography (MI:0114) l MINT-7712032: Ecb2 (uniprotkb:P0A879) and Ecb2 (uniprotkb:P0A879) bind (MI:0407) by biophysical (MI:0013) Abbreviations DSC, differential scanning calorimetry; Eca, tryptophan synthase a subunit from E coli; Ecb, tryptophan synthase monomer b subunit from E coli; Ecb2, tryptophan synthase b2 subunit from E coli; PLP, pyridoxal 5¢-phosphate; Sta, tryptophan synthase a subunit from S typhimurium; Sta2b2, tryptophan synthase a2b2 complex from S typhimurium; Stb, tryptophan synthase monomer b subunit from S typhimurium; Stb2, tryptophan synthase b2 subunit from S typhimurium; Td, denaturation temperature; bA, bB, two b subunits in the same Ecb2 dimer FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS 2157 Apo- and holo-tryptophan synthase b2 subunits K Nishio et al Introduction Tryptophan synthase (EC 4.1.2.20) catalyzes the final two steps in the biosynthesis of l-tryptophan The bacterial enzyme, a multifunctional a2b2 complex (Mr = 143 300), is composed of nonidentical a (Mr = 28 700) and b (Mr = 43 000) subunits The a2b2 complex can be isolated as a monomeric a subunit and dimeric b2 subunits in solution The a and b2 subunits catalyze different reactions, namely the a and b reactions (Eqns 1, respectively) The physiologically important reaction is the ab reaction (Eqn 3), which is catalyzed by the a2b2 complex a reaction: Indole 3-glycerol phosphate $ indole ỵ D-glyceraldehyde 3-phosphate 1ị b reaction: Indole + L-serine ! L-tryptophan ỵ H2 O 2ị ab reaction: Indole 3-glycerol phosphate + L-serine ! L-tryptophan ð3Þ þ D-glyceraldehyde 3-phosphate þ H2 O Combining the a and b2 subunits to form the a2b2 complex stimulates the enzymatic activity of each subunit by one to two orders of magnitude [1,2] This mutual activation of the two subunits is thought to derive from conformational changes in the subunits upon formation of the complex [3,4] Therefore, tryptophan synthase is an excellent model for using to study the relationship between functional activation and conformational changes in proteins The quaternary structure of the a2b2 complex from Salmonella typhimurium (Sta2b2) is an extended linear abba subunit arrangement The active sites of the a ˚ and b subunits of Sta2b2 are connected by a 25–30 A hydrophobic tunnel through which the indole is transferred from the a subunit to the b subunit [5,6] Crystal structures of the Sta2b2 complex with allosteric cations and ⁄ or ligands, and of the Pfa2b2 complex from the hyperthermophile Pyrococcus furiosus, have been described [7–16] These structures provide valuable information to help us understand the allosteric mechanism of the tryptophan synthase Recently, structures have been solved of the tryptophan synthase a subunit from Escherichia coli (Eca) [17] and of the tryptophan synthase a [18], and b2 [19] subunits from P furiosus To obtain a clear understanding of the 2158 reason for a low enzymatic activity of the b2 subunit in the absence of the a subunit, it is necessary to solve the structures of the b2 subunit from E coli (Ecb2) with and without its cofactor, pyridoxal-5¢-phosphate (PLP) PLP-dependent enzymes catalyze multiple reactions during the metabolism of amino acids These enzymes have been classified into a, b and c families based on the chemical characteristics of the enzymatic reactions [20] The tryptophan synthase b2 subunit belongs to the b family, members of which catalyze b-replacement or b-elimination reactions This family has been distinctly classified into five-fold-types based on sequence and structural features [21] Fold-type II enzymes in the b family include the tryptophan synthase b2 subunit, O-acetylserine sulfhydrylase [22] and serine dehydratase [23] Several crystal structures of the apo- and holo-forms of PLP-binding enzymes exhibit only minor re-arrangements in the positions of residues lining the active site between the apo- and holo-enzymes [24,25] Other PLP-binding enzymes, however, display significant conformational changes between the apo- and holo-forms [23,26,27] Both the apo- and holo-types of serine dehydratase, isolated from the rat liver, form a homodimer In the aposerine dehydratase dimeric form, a small domain inserts into the catalytic cleft of the partner subunit so that the active site is closed when inactive Tryptophan synthase, however, is a unique PLP-binding protein because the b reaction mediated by the b subunit is regulated via an allosteric mechanism triggered by association with the cognate a subunit PLP binds cooperatively to the apo-b2 subunit and noncooperatively to the a2apo-b2 complex in E coli [28,29] Therefore, it is important to determine the b2 subunit structure of the apo-type without PLP, as well as that of the holo-type, to elucidate the mechanism of PLP binding We obtained crystals of the apo-form in the absence of PLP, as well as the holo-type bearing PLP, for Ecb2 The structures of the apo-b2 and holo-b2 ˚ subunits were solved at 3.0 and 2.9 A resolutions, respectively We evaluated the thermal stabilities of the holo-Ecb2 and apo-Ecb2 forms using differential scanning calorimetry (DSC) In this communication we will discuss the role of PLP in the stabilization of Ecb2, the mechanism of PLP binding to apo-Ecb2 and the structural basis for lower enzymatic activity of Ecb2 in the absence of the a subunit, from a comparison of the apo-Ecb2, holo-Ecb2 and Sta2b2 complex structures FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS Apo- and holo-tryptophan synthase b2 subunits K Nishio et al Results and Discussion Contribution of PLP to the stabilization of the b2 subunit Using the holo-Ecb2, the apo-Ecb2 and the reconstituted holo-Ecb2 proteins, we confirmed the contribution of PLP to the stabilization of the b2 subunits Holo-Ecb2 bound to PLP demonstrated an absorption spectrum bearing a peak at 413 nm, characteristic for a PLP internal aldimine bound to a Lys in the b2 subunit [30], and a CD spectrum with a peak around 420 nm (data not shown) Dissociation of PLP from the b2 subunit by dialysis against 0.1 m Mes buffer (pH 6.5) containing 0.1 m Li2SO4 could be confirmed by the disappearance of these peaks from repeat analyses Ultracentrifugation analysis indicated that both apo-Ecb2 and holo-Ecb2 remain in dimeric forms in solution Reconstitution of holo-Ecb2 from apo-Ecb2 by dialysis against 50 mm potassium phosphate buffer (pH 7.0) containing 0.2 mm PLP for day at °C was also confirmed by the characteristic changes seen on absorption and CD spectra, and DSC curves Figure displays the pH dependence of holo-Ecb2 and apo-Ecb2 stabilities; the denaturation temperature, Td, of the holo-protein increases by nearly 25 °C at approximately pH as a result of PLP binding The difference in the Td values between the holo- and apoproteins decreased with decreasing pH (Fig 1), suggesting that the binding constant of PLP decreases at pH 6.5 The denaturation temperature of the reconstituted holo-Ecb2 was similar to that of the native holo-Ecb2 bound to PLP (Fig 1), indicating that the dissociation of PLP from holo-Ecb2 is reversible These Fig pH dependence of the thermal stability (Td) of holo-Ecb2 and apo-Ecb2 The Td value represents the peak temperature on the DSC curve measured at a scan rate of °CỈmin)1 Closed circles, open circles, and open triangles indicate the Td values for the native holo-, reconstituted holo- and apo-b2 subunits, respectively Buffers used were 50 mM potassium phosphate supplemented with mM EDTA at a pH of