Binding synergy and cooperativity in dihydrodipicolinate reductase: implications for mechanism and the design of biligand inhibitors

Biochemistry
Xia GeDaniel S Sem

Abstract

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...Continue Reading

References

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Citations

Dec 1, 2017·The Biochemical Journal·Serena A J WatkinF Grant Pearce
Aug 11, 2018·Biochemical and Biophysical Research Communications·Swanandi PoteMaksymilian Chruszcz

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