Structural Characterization of the Reaction Pathway in Phosphoserine Phosphatase: Crystallographic “snapshots” of Intermediate States Weiru Wang 1 , Ho S. Cho 2 , Rosalind Kim 2 , Jaru Jancarik 1 Hisao Yokota 2 , Henry H. Nguyen 2 , Igor V. Grigoriev 1 David E. Wemmer 1,2 * and Sung-Hou Kim 1,2 * 1 Department of Chemistry University of California Berkeley, CA 94720-5230, USA 2 Physical Biosciences Division of the Lawrence Berkeley National Laboratory, Berkeley CA 94720, USA Phosphoserine phosphatase (PSP) is a member of a large class of enzymes that catalyze phosphoester hydrolysis using a phosphoaspartate – enzyme intermediate. PSP is a likely regulator of the steady-state D-serine level in the brain, which is a critical co-agonist of the N-methyl-D-aspartate type of glutamate receptors. Here, we present high-resolution (1.5 – 1.9 A ˚ ) struc- tures of PSP from Methanococcus jannaschii, which define the open state prior to substrate binding, the complex with phosphoserine substrate bound (with a D to N mutation in the active site), and the complex with AlF 3 , a transition-state analog for the phospho-transfer steps in the reac- tion. These structures, together with those described for the BeF 3 2 complex (mimicking the phospho-enzyme) and the enzyme with phosphate product in the active site, provide a detailed structural picture of the full reaction cycle. The structure of the apo state indicates partial unfolding of the enzyme to allow substrate binding, with refolding in the presence of substrate to provide specificity. Interdomain and active-site confor- mational changes are identified. The structure with the transition state analog bound indicates a “tight” intermediate. A striking structure homology, with significant sequence conservation, among PSP, P-type ATPases and response regulators suggests that the knowledge of the PSP reaction mechanism from the structures determined will provide insights into the reaction mechanisms of the other enzymes in this family. q 2002 Elsevier Science Ltd. All rights reserved Keywords: phosphotransfer, phospho-monoester hydrolysis; phospho- aspartyl enzyme intermediate; phosphoserine phosphatase; associative mechanism; dissociative mechanism *Corresponding authors Introduction Reversible protein phosphorylation plays a cen- tral role in regulating basic functions in eukaryotic processes such as DNA replication, 1 cell cycle control, 2,3 gene transcription, 4 protein translation, 5 and energy metabolism. Protein phosphorylation is required also for more complex functions in higher eukaryotes, such as cell, organ, and limb differentiation, 6 cell survival, synaptic trans- mission, 7 cell-substratum and cell – cell communi- cation, 8 and to mediate complex interactions with the external environment. Formation and breakage of phosphate monoester bonds are key steps in regulating protein activity. Over the past four decades, much research effort has been devoted to elucidation of the reaction mechanisms of phosphate monoester hydrolysis, but there has been substantial debate focused on the nature of the transition-state. 9–12 Figure 1 illus- trates the two extreme mechanisms proposed; the unimolecular decomposition or dissociative mechanism in which the transition state is thought to be a metaphosphate-like species, and the bimolecular association or associative mechanism 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail addresses of the corresponding authors: dewemmer@lbl.gov; shkim@cchem.berkeley.edu Abbreviations used: PSP, phosphoserine phosphatase; PTP, phosphotyrosine phosphatase; PLS, phospho-L- serine; APUP, aspartyl-phosphate-utilizing phospho- hydrolase/phosphotransferase; HAD, haloacid dehalogenase-like hydrolase; NMDA, N-methyl-D- aspartate. doi:10.1016/S0022-2836(02)00324-8 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 319, 421–431