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The structure of mouse L 1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0/~ resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

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The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 'H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase.`3C relaxation measurements reveal dihedral angle changes of ± 25°to ± 350 on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least ± 65°o n the millisecond time scale. 'H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.A knowledge of the internal motions of proteins is of considerable importance for understanding their structurefunction relationships (1)(2)(3)(4). In recent years, a variety of theoretical (4-6) and experimental (3, 7-14) methods have been brought to bear on this problem, and a picture of the kinds of motion that take place is beginning to emerge. Hitherto, most attention has been focused on the atoms of the protein itself, although similar dynamic behavior would be expected for small molecules bound to proteins, and this has indeed been observed in a few cases (10, 15-18). We have been studying the binding of the antibacterial drug trimethoprim ( Fig. 1 (26,27), with the spectral density function J(w) = 5 'TR_ + (1 -S2)T )[2]The internal motion is characterized by two parameters: Tint, an effective correlation time, and S2, the square of the order parameter for the motion.

PRINTS is a compendium of protein motif fingerprints derived from the OWL composite sequence database. Fingerprints are groups of motifs within sequence alignments whose conserved nature allows them to be used as signatures of family membership. Fingerprints inherently offer improved diagnostic reliability over single motif methods by virtue of the mutual context provided by motif neighbors. To date, 650 fingerprints have been constructed and stored in PRINTS, the size of which has doubled in the last 2 years. The current version, 14.0, encodes 3500 motifs, covering a range of globular and membrane proteins, modular polypeptides, and so on. The database is now accessible via the UCL Bioinformatics Server on http:@ www.biochem.ucl.ac.uk/bsm/dbbrowser/. We describe here progress with the database, its compilation and interrogation software, and its Web interface.