Dynamics of trimethoprim bound to dihydrofolate reductase--a deuterium NMR study.

We have employed deuterium NMR techniques to determine the dynamics of trimethoprim (TMP) in a binary complex with dihydrofolate reductase (DHFR) or in a ternary complex with DHFR and cofactor NADP+ in the fully hydrated state. TMP was deuterated at the following positions: (2',6'-D2)TMP, (3'-Ome-D3)TMP and (3',4'-Ome-D6)TMP. Dynamics of TMP were deduced from lineshape simulation and relaxation measurements of the deuterium NMR powder spectra of the three samples obtained at various temperatures. The results showed that in the polycrystalline state the TMP molecule is very rigid. The only detectable motion is the methyl group rotation at a rate of 10(10) s-1 at 25 degrees C, as determined from simulation of the partially relaxed powder patterns. When bound to DHFR a residual deuterium quadrupole splitting of 140 kHz was observed for (2',6'-D2)TMP at temperatures up to 30 degrees C, suggesting that the benzyl ring in the bound state is also very rigid. In contrast, in the binary complex with DHFR the methoxyl groups of TMP undergo librational motion of 10(7) s-1 about the C3-O bond at an amplitude of 54 degrees for the meta methoxyl group and about the C4-O bond at an amplitude of 70 degrees and similar rate for the para methoxyl group at 30 degrees C. The presence of the cofactor, NADP+, appears to tighten up the binding pocket such that the motion freedom of TMP is more restricted. The rigidity of TMP in a protein complex as revealed by our deuterium NMR results is in accord with the tight binding of TMP to DHFR.