Valentina Goldfarb

Aliphatic 1H and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy

SummaryExtensive 1H and 13C assignments have been obtained for the aliphatic resonances of a uniformly 13C-and 15N-labeled recombinant VL domain from the anti-digoxin antibody 26-10. Four-dimensional triple resonance NMR data acquired with the HNCAHA and HN(CO)CAHA pulse sequences [Kay et al. (1992) J. Magn. Reson., 98, 443–450] afforded assignments for the backbone HN, N, Hα and Cα resonances. These data confirm and extend HN, N and Hα assignments derived previously from three-dimensional 1H-15N NMR studies of uniformly 15N-labeled VL domain [Constantine et al. (1992), Biochemistry, 31, 5033–5043]. The identified Hα and Cα resonances provided a starting point for assigning the side-chain aliphatic 1H and 13C resonances using three-dimensional HCCH-COSY and HCCH-TOCSY experiments [Clore et al. (1990), Biochemistry, 29, 8172–8184]. The Cα and Cβ chemical shifts are correlated with the VL domain secondary structure. The extensive set of side-chain assignments obtained will allow a detailed comparison to be made between the solution structure of the isolated VL domain and the X-ray structure of the VL domain within the 26–10 Fab.

Design, synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: Observation of extensive dynamics in an enzyme/inhibitor complex.

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11μM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg(2+). In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca(2+) and Mg(2+) are indicative of a highly dynamic process in the active site for the HDHD4/Mg(2+)/3 complex. Possible explanations for this observation are discussed.

Cloning, purification, crystallization and preliminary X-ray analysis of the catalytic domain of human receptor-like protein tyrosine phosphatase γ in three different crystal forms.

Protein tyrosine phosphatase γ is a membrane-bound receptor and is designated RPTPγ. RPTPγ and two mutants, RPTPγ(V948I, S970T) and RPTPγ(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.

Structural and functional characterization of CFE88: Evidence that a conserved and essential bacterial protein is a methyltransferase

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227‐residue protein has minimal sequence similarity to proteins of known 3Dstructure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side‐chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N‐terminal domain with 163 residues and a C‐terminal domain with 64 residues. The C‐terminal domain is primarily helical, while the N‐terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X‐ray structure of ErmC′ methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S‐adenosyl‐L‐homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N‐terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.