Binding synergy and cooperativity in dihydrodipicolinate reductase: implications for mechanism and the design of biligand inhibitors.
Dihydrodipicolinate reductase (DHPR) is a homotetramer that catalyzes reduction of dihydrodipicolinate (DHP). We recently reported a biligand inhibitor ( K i = 100 nM) of DHPR, comprised of fragments that bind in the NADH (CRAA = catechol rhodanine acetic acid) and DHP (PDC = pyridine dicarboxylate) binding sites. Herein, we characterize binding synergy and cooperativity for ligand binding to Escherichia coli DHPR: NADH or CRAA and PDC (stable analog of DHP). While K d values indicate little synergy between NADH and PDC, (1)H- (15)N HSQC chemical shift perturbation and saturation transfer difference (STD) titrations indicate that PDC induces a more dramatic conformational change than NADH, consistent with a role in domain closure. PDC binds cooperatively (Hill coefficient = 2), while NADH does not, based on STD titrations that monitor only fast exchange processes. However, HSQC titrations monitoring Trp253 (located between monomers) indicate that NADH binds in two steps, with high affinity binding to only one of the monomers. Therefore, DHPR binds cofactor via a sequential model, with negative cooperativity. These results, interpreted in light of steady-state data, suggest that DHPR activity requires NADH binding at only one of the four monomers. Implications of our results for fragment assembly are discussed, using CRAA tethering to PDC as a model biligand: (a) if one fragment (ex. PDC) must induce a large structural change before the other fragment is brought proximal, this must be screened for upfront, and (b) cooperative or synergistic interactions between binding sites can lead to unexpected and misleading effects in NMR-based screening.
Ge
Xia
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Chemical Proteomics Facility at Marquette, Department of Chemistry, P.O. Box 1881, Marquette University, Milwaukee, Wisconsin 53201, USA.
Olson
Andrew
A
Cai
Sheng
S
Sem
Daniel S
DS
D001665:Binding Sites / Q000502:physiology; D050746:Dihydrodipicolinate Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D013379:Substrate Specificity / Q000502:physiology
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D008024:Ligands; D050746:Dihydrodipicolinate Reductase