letters nature structural biology • volume 8 number 11 • november 2001 947 Delineation of the allosteric mechanism of a cytidylyl- transferase exhibiting negative cooperativity Shawn Y. Stevens 1 , Subramaniam Sanker 2,3 , Claudia Kent 2 and Erik R.P. Zuiderweg 1,2,4 1 Biophysics Research Division, University of Michigan, 930 North University, Ann Arbor, Michigan 48109-1055, USA. 2 Department of Biochemistry, University of Michigan, 4417 Medical Sciences Building I, Ann Arbor, Michigan 48109-0606, USA. Present address: 9500 Euclid Avenue, Cleveland Clinic Foundation, Department of Molecular biology, NC2-119, Cleveland, Ohio 44195, USA. 4 Department of Chemistry, University of Michigan, 930 North University, Ann Arbor, Michigan 48109-1055, USA. The dimeric enzyme CTP:glycerol-3-phosphate cytidylyl- transferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indi- cate an extensive loss of conformational exchange broaden- ing of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin. Cooperative binding processes are widespread in Nature and are important for regulation of key enzymes and receptors. In general, cooperativity is controlled by an allosteric response to ligand binding that changes the affinity for subsequent binding events. One of the earliest goals in structural biology has been to understand allosteric mechanisms, but obtaining structural information on the intermediates in the cooperative processes has often been difficult. This is particularly true for systems that display positive cooperativity, which appear to be more prevalent in Nature 1 . Less common are examples of negative cooperativity, but the decrease in affinity for successive ligands can make these systems amenable to structural studies of naturally occurring ligand-bound intermediates and, hence, allow a detailed under- standing of the allosteric mechanism. The enzyme glycerol-3-phosphate:CTP transferase (GCT) is an example of a system that displays strong negative cooperativi- ty. The enzyme is a 28 kDa homodimer that catalyzes the synthe- sis of CTP and glycerol-3-phosphate to CDP–glycerol, a precursor to teichoic acid, which is a primary component in the synthesis of cell walls in many bacteria 2,3 . The two substrates bind at different loci, with magnesium required for glycerol-3- phosphate binding. For catalysis to occur, all four substrate bind- ing sites of GCT must be filled. The affinity between the first and fourth substrate binding events decreases by several orders of magnitude 4 (Fig. 1a). The crystal structure of the GCT homodimer with two bound CTP molecules provides a picture of ligand recognition involving a complex set of interactions 5 . In particular, the 113-RTEGISTT loop wraps around the nucleotide substrate to form a network of interactions that provides much of the specificity of the enzyme. The crystal structure indicates that this loop, in addition to other residues, must move in order to accommodate the ligand (Fig. 1b). Although this suggests a possible action for the allosteric mechanism, insight from a single static structure does not provide a complete under- standing of the catalytic activity of GCT or the allosteric mechanism that governs this activity. Here we report the use of NMR spectroscopy to further elucidate the allosteric mecha- nism by characterizing individual states of the GCT enzyme leading to saturation with CTP. Monitoring substrate binding with Arg side chains Arg 55, Arg 63 and Arg 113 had been identified as important for catalytic activity 5,6 . In the crystal structure, these Arg residues were shown to play an important role in CTP recognition as well as communication across the dimer interface 5 . The loop con- taining Arg 113 (113-RTGESITT) is highly conserved within the cytidylyltransferase family and establishes a complex set of inter- actions with the ligand (Fig. 1b). The side chain of Arg 63 is far removed from the CTP binding site and extends across the dimer interface to the opposite subunit (Fig. 2a). In the unligated form, only the 1 Hε- 15 Nε 2D NMR cross peaks 7 of Arg 63 and Arg 113 are observed (Fig. 2b), with the guanido protons of Arg 23 and Arg 55 exchanging too rapidly CTP CTP CTP CTP CTP CTP CTP CTP CTP CTP G3P G3P G3P G3P G3P G3P G3P G3P G3P G3P Mg 2+ 1 300 300 4000 1 300 300 a b Fig. 1 Structure and mechanism of GCT. a, The proposed mechanism of GCT is a random-order kinetics model. All four substrates must be pre- sent for catalytic activity to be observed. Relative dissociation constants are depicted 4,6 . The structures in brackets indicate potential asymmetric ligation pathways (with unknown affinities), connecting states with one and three ligands. b, The crystal structure of GCT has been determined 5 to 2.0 Å resolution, showing the GCT homodimer complexed with a CTP molecule (purple) in each active site. Residues important for activity are labeled 6 . Three regions well conserved in the cytidylyltransferase family are labeled in black. Site-directed mutagenesis revealed that mutation of individual residues in all three sequences is detrimental to activity of GCT 5,6 . (b) was made using MOLMOL 28 . © 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com