Keith L. Constantine

Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28.

The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99 YPPPY104 motif are adjacent to a patch of charged surface residues on the A‘GFCC’ face of the protein. Mutation of these residues, which are conserved in the CTLA-4/CD28 family, significantly reduces binding to CD80 and/or CD86, implicating this patch as a ligand binding site.

Redefining the minimal antigen-binding fragment.

The X-ray structures of a lysozyme-complexed camel VH domain and an antigen-free llama VH domain reveal novel features that open up new possibilities for generating and engineering minimal antigen recognition units.

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

SummaryThe 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys1 with Cys13 and Cys7 with Cys19, and the side chain of Asp9 forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H2O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.

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.

Evaluation of site-directed spin labeling for characterizing protein-ligand complexes using simulated restraints.

Simulation studies have been performed to evaluate the utility of site-directed spin labeling for determining the structures of protein-ligand complexes, given a known protein structure. Two protein-ligand complexes were used as model systems for these studies: a 1.9-A-resolution x-ray structure of a dihydrofolate reductase mutant complexed with methotrexate, and a 1.5-A-resolution x-ray structure of the V-Src tyrosine kinase SH2 domain complexed with a five-residue phosphopeptide. Nitroxide spin labels were modeled at five dihydrofolate reductase residue positions and at four SH2 domain residue positions. For both systems, after energy minimization, conformational ensembles of the spin-labeled residues were generated by simulated annealing while holding the remainder of the protein-ligand complex fixed. Effective distances, simulating those that could be obtained from (1)H-NMR relaxation measurements, were calculated between ligand protons and the spin labels. These were converted to restraints with several different levels of precision. Restrained simulated annealing calculations were then performed with the aim of reproducing target ligand-binding modes. The effects of incorporating a few supplementary short-range (< or =5.0 A) distance restraints were also examined. For the dihydrofolate reductase-methotrexate complex, the ligand-binding mode was reproduced reasonably well using relatively tight spin-label restraints, but methotrexate was poorly localized using loose spin-label restraints. Short-range and spin-label restraints proved to be complementary. For the SH2 domain-phosphopeptide complex without the short-range restraints, the peptide did not localize to the correct depth in the binding groove; nevertheless, the orientation and internal conformation of the peptide was reproduced moderately well. Use of the spin-label restraints in conjunction with the short-range restraints resulted in relatively well defined structural ensembles. These results indicate that restraints derived from site-directed spin labeling can contribute significantly to defining the orientations and conformations of bound ligands. Accurate ligand localization appears to require either a few supplementary short-range distance restraints, or relatively tight spin-label restraints, with at least one spin label positioned so that some of the restraints draw the ligand into the binding pocket in the latter case.

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.

Relaxation study of the backbone dynamics of human profilin by two‐dimensional 1H‐15N NMR

The dynamic properties of 111 backbone HN sites in uncomplexed human profilin, a protein of 139 residues, have been characterized by two‐dimensional inverse‐detected 1H‐15N NMR spectroscopy. Heteronuclear {1H}‐15N nuclear Overhauser effects and 15N longitudinal and transverse relaxation rates have been analyzed in terms of model‐free spectral density functions and exchange contributions to transverse relaxation rates. Relatively high mobilities on the nanosecond timescale are observed for Asp26 and Ser27, which form part of a loop connecting β‐strands A and B, and for Thr92 through Ala95, which are in a loop connecting β‐strands E and F. Significant exchange contributions, indicative of motions on the microsecond to millisecond timescale, have been obtained for 30 residues. These include Leu77, Asp80 and Gly81 of a loop between β‐strands D and E, Ser84 and Met85 of β‐strand E, Gly121 of a loop connecting β‐strand G and the C‐terminal helix, and Gln138, which is next to the C‐terminal residue Tyr139. Some of the regions showing high flexibility in profilin are known to be involved in poly‐L‐proline binding.

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.