Bacillus stearothermophilus PFK (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose 6-phosphate (Fru-6-P), binds along the other dimer-dimer interface. Evans et al., observed that the inhibitor, phosphoglycolate, bound structure, when compared to the substrate and activator bound structure of wild-type BsPFK, exhibits a 7° rotation about the substrate-binding interface, termed the quaternary shift [Schirmer, T., and Evans, P. R. (1990) Nature 343, 140-145]. We report that the variant D12A BsPFK exhibits a 100-fold increase in the binding affinity for PEP, a 50-fold decrease in the binding affinity for Fru-6-P, but an inhibitory coupling comparable to wild type. Crystal structures of the apo and PEP bound forms of D12A BsPFK have been determined (Protein Data Bank ID codes 4I36 and 4I7E, respectively), and both indicate a shifted structure similar to the inhibitor-bound structure of wild type. D12 does not directly bind to either substrate or inhibitor and is located along the substrate-binding interface. A conserved hydrogen bond between D12 and T156 forms across the substrate-binding subunit-subunit interface in the substrate-bound form of BsPFK. The variant T156A BsPFK, when compared to wild-type, shows a 30-fold increase in PEP binding affinity, a 17-fold decrease in Fru-6-P binding affinity, and an estimated coupling that is also approximately equal to wild-type. In addition, the T156A BsPFK crystal structure bound to PEP is reported (Protein Data Bank ID code 4I4I), and it exhibits a shifted structure similar to D12A BsPFK and the inhibitor-bound structure of wild type. The results suggest that main role of the quaternary shift may be to influence ligand binding and not to cause the heterotropic allosteric inhibition per se.

The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was solved using molecular replacement to 2.8 Å resolution (PDB ID code 3U39). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produce is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose 6-phosphate (Fru-6-P), is allosterically regulated by the K-type inhibitor phosphoenolpyruvate (PEP). The allosteric coupling between the substrate and inhibitor is quantified by a standard coupling free energy that defines an equilibrium with the Fru-6-P-bound and PEPbound complexes on one side and the apo form and ternary complex on the other. Methyl-transverse relaxation-optimized spectroscopy (Me-TROSY) nuclear magnetic resonance was employed to gain structural information about BsPFK in all four states of ligation relevant to the allosteric coupling. BsPFK was uniformly labeled with 15 N and 2 H and specifically labeled with δ-[ 13 CH 3 ]-isoleucine utilizing an isotopically labeled α-keto acid isoleucine precursor. Me-TROSY experiments were conducted on all four ligation states, and all 30 isoleucines, which are well dispersed throughout each subunit of the enzyme, are well-resolved in chemical shift correlation maps of 13 C and 1 H. Assignments for 17 isoleucines were determined through three-dimensional HMQC-NOESY experiments with [U-15 N, 2 H];Ileδ1-[ 13 CH 3 ]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2 H, 15 N, 13 C]-BsPFK. The assignments allowed for the mapping of resonances representing isoleucine residues to a previously determined X-ray crystallography structure. This analysis, performed for all four states of ligation, has allowed specific regions of the enzyme influenced by the binding of allosteric ligands and those involved in the propagation of the allosteric effect to be identified and distinguished from one another.

Small ankyrin-1 (sAnk1, Ank1.5) is a splice variant of the ANK1 gene that binds to the large modular protein, obscurin A, with nanomolar affinity, a reaction that may help to organize the sarcoplasmic reticulum in striated muscle. A subset of lysine and arginine residues in the 2 ankyrin repeats of sAnk1 interact specifically with 4 glutamate residues in a stretch of 30 amino acids of obscurin to mediate binding. Homology modeling and molecular dynamics simulations have revealed a ''hot spot'' of 4 hydrophobic residues exposed on the surface of the ankyrin repeat domain of sAnk1. We used site-directed mutagenesis of bacterially expressed fusion proteins, followed by blot overlays and surface plasmon resonance assays, to study the contribution of these 4 residues, V70, F71, I102 and I103, to binding to the 30-mer of obscurin. Alanine mutations of each of these four residues inhibited binding to residues 6316-6345 of obscurin (Obsc 6316-6345 ). In contrast, V70A and I102A mutations had no effect on binding to a second sAnk1 binding site on obscurin, located within residues 6231-6260 (Obsc 6231-6260 ). Using the same methods, we mutated the 5 hydrophobic residues present in Obsc 6316-6345 to alanine and identified V6328, I6332, and V6334 as critical for proper binding. Our results suggest that hydrophobic interactions as well as electrostatic interactions are important for the binding of sAnk1 to Obsc 6316-6345 , consistent with studies of the complexes formed by other ankyrin repeat proteins with their ligands. Hydrophobic interactions are likely to contribute to the difference in affinity of sAnk1 for Obsc 6316-6345 and Obsc 6231-6260 , and for the dominant role played by the more C-terminal sequence in binding. Supported by grant R01-AR056330 from the NIH to RJB and training grants T32 GM08181 (to RJB) and T32 AR07592 (to M. Schneider).

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