NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine.
1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Birdsall
B
B
Laboratory of Molecular Structure, National Institute for Medical Research, London, United Kingdom.
Tendler
S J
SJ
Arnold
J R
JR
Feeney
J
J
Griffin
R J
RJ
Carr
M D
MD
Thomas
J A
JA
Roberts
G C
GC
Stevens
M F
MF
D001665:Binding Sites; D005461:Fluorine; D006859:Hydrogen; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011739:Pyrimethamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D005461:Fluorine; D009249:NADP; D006859:Hydrogen; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine