A simple thermodynamic model is formulated for the purpose of interpreting scanning calorimetry data on proteins that have interacting domains. Interactions are quantified by inclusion of an interface free energy, delta GAB, in the thermodynamics of unfolding for multidomain proteins. The assumption is made that delta GAB goes to zero with the unfolding of either domain involved in pairwise interaction, so the interaction term appears to stabilize only the domain with the lower TM. Application of the model to calorimetric data leads to an estimate of -25,000 cal/mol for interactions between the regulatory and catalytic subunits of native aspartate transcarbamoylase and to a value of 0 for delta GAB between the transmembrane and cytoplasmic domains of band 3 of the human erythrocyte membrane. Estimates of changes in delta GAB are also obtained for mutant forms of yeast phosphoglycerate kinase that have been altered in the hinge region between amino-terminal and carboxy-terminal domains. The model is also applied to ligand binding to proteins having domains that communicate through pairwise interaction. It is shown that whenever the delta GAB term is ligand-dependent, then attachment of the ligand to the binding domain will be partially controlled by the other (regulatory) domain. This situation can sometimes be recognized and quantified when calorimetric scans are carried out at varying ligand concentrations. According to the model, the binding of MgATP to the carboxy-terminal domain of phosphoglycerate kinase is strongly stabilized (ca. 20% of the unitary free energy of binding) by participation of the amino-terminal domain, which acts to increase the binding constant 25-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoglycerate kinase (PGK) catalyzes a reversible transfer of a phosphoryl group from 1,3-bisphosphoglycerate to ADP in the glycolytic pathway. The crystal structures of the substrate-free enzyme and its binary complex with ATP have been determined for horse muscle (4) and yeast (5) PGKs and represent an "open" conformation. The distance between the y-phosphate of ATP bound to the C-terminal domain is >10 A away from the 3-phosphoglycerate (3-PG) binding site situated on the N-terminal domain. This distance is reduced to -7.2 A in the binary complex ofpig muscle PGK with 3-PG (6). This decrease and an accompanying domain movement consisting ofa 7.7°rotation ofthe two domains are smaller than predicted for the ternary complex (1, 7).Evidence for a substrate-induced conformational change in PGK has been obtained from small-angle x-ray scattering experiments in solution that demonstrated a decrease in the radius of gyration (Rg) of 1 A for the ternary complex with . This decrease was found to be consistent with a theoretical model that assumes a hinge-bending rigid body domain motion (8). Site-directed mutagenesis experiments suggested the importance of the structural elements of the hinge region for the transmission of substrateinduced conformational changes in yeast . Although they provided additional support for the hingebending hypothesis, the methods used in these studies were not capable ofcharacterizing the dynamics ofconformational fluctuations of PGK in solution.In this paper we have combined genetic engineering to create specific labeling sites for fluorescent probes and time-resolved fluorescence energy transfer measurements (14-17) to study the conformational flexibility of yeast PGK. This manuscript describes a set of experiments designed to probe the equilibrium conformations of PGK in solution and the effect of added substrates on the conformational state of the enzyme.

The structure of a ternary complex of the R65Q mutant of yeast 3-phosphoglycerate kinase (PGK) with magnesium 5'-adenylylimidodiphosphate (Mg-AMP-PNP) and 3-phospho-D-glycerate (3-PG) has been determined by X-ray crystallography to 2.4 angstrom resolution. The structure was solved by single isomorphous replacement, anamalous scattering, and solvent flattening and has been refined to an R-factor of 0.185, with rms deviations from ideal bond distance and angles of 0.009 angstrom and 1.78 degrees, respectively. PGK consists of two domains, with the 3-PG bound to a "basic patch" of residues from the N-terminal domain and the Mg-AMP-PNP interacting with residues from the C-terminal domain. The two ligands are separated by approximately 11 angstrom across the interdomain cleft. The model of the R65Q mutant of yeast PGK is very similar to the structures of PGK isolated from horse, pig, and Bacillus stearothermophilus (rms deviations between equivalent alpha-carbons in the individual domains < 1.0 angstrom) but exhibits substantial variations with a previously reported yeast structure (rms deviations between equivalent alpha-carbons in the individual domains of 2.9-3.2 angstrom). The most significant tertiary structural differences among the yeast R65Q, equine, porcine, and B. stearothermophilus PGK structures occur in the relative orientations of the two domains. However, the relationships between the observed conformations of PGK are inconsistent with a "hinge-bending" behavior that would close the interdomain cleft. It is proposed that the available structural and biochemical data on PGK may indicate that the basic patch primarily represents the site of anion activation and not the catalytically active binding site for 3-PG.