Biomedical subjects

A H Gunasekera

Publications and source records attributed to A H Gunasekera.

8 recordsLinked to original sources

PubMed2001

Solution structure and function of a conserved protein SP14.3 encoded by an essential Streptococcus pneumoniae gene.

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity rates and has developed resistance to many antibiotics. The genome of S. pneumoniae has recently been completely sequenced revealing many genes encoding hypothetical proteins of unknown function. We have found that the gene encoding one such conserved protein, SP14.3, is essential for growth of S. pneumonia. Since it is essential, SP14.3 represents a potential target for drug discovery. Here, we describe the three-dimensional solution structure of SP14.3 as determined by NMR spectroscopy. The structure consists of two domains each with an alpha/beta-fold. The N-terminal domain contains two alpha-helices and a three-stranded beta-sheet, while the C-terminal domain is composed of one alpha-helix and a five-stranded beta-sheet. The N-terminal domain of the protein contains a highly negatively charged surface and resembles the fold of the N-terminal domain of Thermus thermophilus ribosomal protein S3. The C-terminal domain has a protein fold similar to human small nuclear ribonucleoprotein Sm D3 and Haloarcula marismortui ribosomal protein L21E. The two domains of the protein tumble in solution overall as a whole with an overall molecular rotational correlation time (tau(m)) of 12.9 ns at 25 degrees C. The relative orientation of the two domains is not defined by the nuclear Overhauser effect data. Indeed, residual dipolar couplings and the structure calculations indicate that the relative orientation of the two domains is not rigidly oriented with respect to one another in solution.

Bacterial Proteins↗

PubMed2000

NMR structure and mutagenesis of the third Bir domain of the inhibitor of apoptosis protein XIAP.

The inhibitor of apoptosis proteins (IAPs) regulate the caspase family of cysteine proteases, which play an important role in the execution of programmed cell death. Human X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases-3, -7, and -9. Here we show that the Bir3 domain is the minimal region of XIAP that is needed for potent caspase-9 inhibition. The three-dimensional structure of the Bir3 domain of XIAP, determined by NMR spectroscopy, resembles a classical zinc finger and consists of five alpha-helices, a three-stranded beta-sheet, and a zinc atom chelated to three cysteines and one histidine. The structure of the Bir3 domain is similar to that of the Bir2 domain of XIAP but differs from the previously determined structure of the Bir3 domain of MIHB. Based on site-directed mutagenesis, we have identified the regions of the Bir3 domain of XIAP that are important for inhibiting caspase-9. Despite the structural similarities of the Bir2 and Bir3 domain of XIAP, a different set of residues were found to be critical for inhibiting the individual caspases. These results suggest that XIAP inhibits caspase-3 and caspase-9 in a different manner.

Amino Acid Sequence↗

PubMed2000

Expression, refolding, and isotopic labeling of human serum albumin domains for NMR spectroscopy.

Many different compounds bind to human serum albumin (HSA), which can be a significant problem in the drug discovery process. To aid in the design of drug molecules that do not bind to HSA, the structures of HSA/ligand complexes would be very useful. However, little information has been reported on the structures of small molecules complexed to HSA. In this paper, we describe a procedure for preparing isotopically labeled domains of HSA for nuclear magnetic resonance (NMR) studies. The procedure involves the expression in Escherichia coli, refolding, and a multistep purification. Domains I and III are capable of folding into stable structural units and producing well resolved (15)N/(1)H correlation spectra, whereas domain II forms significant aggregates at sub-millimolar concentration. Using our protocols, isotopically labeled and properly folded domains I and III can be effectively produced in large quantities for NMR-based structural studies and NMR-based screening. This provides a valuable tool for obtaining structural information on HSA/ligand complexes by NMR which will be useful in drug discovery.

Cloning, Molecular↗

PubMed1999

NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP.

The inhibitor-of-apoptosis (IAP) family of proteins, originally identified in baculoviruses, regulate programmed cell death in a variety of organisms. IAPs inhibit specific enzymes (caspases) in the death cascade and contain one to three modules of a common 70-amino-acid motif called the BIR domain. Here we describe the nuclear magnetic resonance structure of a region encompassing the second BIR domain (BIR2) of a human IAP family member, XIAP (also called hILP or MIHA). The structure of the BIR domain consists of a three-stranded antiparallel beta-sheet and four alpha-helices and resembles a classical zinc finger. Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were found to be critical for inhibiting caspase-3. The absence or presence of these residues may explain the differences in caspase inhibition observed for different truncated and full-length IAPs. Our data further indicate that these residues may bind to the active site and that the BIR domain may interact with an adjacent site on the enzyme.

Amino Acid Sequence↗

PubMed1999

Accurate measurement of H(N)-H(alpha) residual dipolar couplings in proteins.

A method for accurately measuring H(N)-H(alpha) residual dipolar couplings is described. Using this technique, both the sign and magnitude of the coupling can be determined easily. Residual dipolar coupling between H(N)(i)-H(alpha)(i) and H(N)(i)-H(alpha)(i-1) were measured for the FK506 binding protein complexed to FK506. The experimental values were in excellent agreement with predictions based on an X-ray crystal structure of the protein/ligand complex, suggesting that these residual dipolar couplings will provide accurate structural constraints for the refinement of protein structures determined by NMR.

Magnetic Resonance Spectroscopy↗

PubMed1996

Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds. No differences are observed for the alpha subunit.

Amino Acid Sequence↗

PubMed1996

Lymphoma-selective antibody Lym-1 recognizes a discontinuous epitope on the light chain of HLA-DR10.

The selectivity of Lym-1 for malignant B lymphocytes makes this monoclonal antibody a promising candidate for the delivery of toxic agents to malignant B cells. The original immunogen used for the development of Lym-1 was Raji Burkitt's lymphoma cell nuclei [Epstein A. L., Marder R. J., Winter J. N., Stathopoulos E., Chen F. M., Parker J. W., Taylor C. R. (1987) Cancer Res 47: 830]. The Lym-1 antigen was characterized at that time as a polymorphic HLA-DR variant. We prepared an affinity column using immobilized Lym-1 to isolate the Lym-1 antigen from Raji cell lysate. Immunological characterization of the immunoaffinity-purified Lym-1 antigen on Western blots led to the conclusion that the antigen is the beta chain of HLA-DR10. This was confirmed by Edman sequencing of the isolated polypeptide chain. Western blots further show that the Lym-1 epitope is only recognized if the beta chain disulfide bonds are intact. These results imply that Lym-1 binds a discontinuous epitope on the beta chain of HLA-DR10.

Amino Acid Sequence↗

PubMedDate not supplied

Immunoassay reagents for thyroid testing. 2. Binding properties and energetic parameters of a T4 monoclonal antibody and its Fab fragment with a library of thyroxine analog biosensors using surface plasmon resonance.

A library of 16 thyroxine analog biosensors were prepared for use on the BIAcore surface plasmon resonance instrument. An anti-thyroxine monoclonal antibody and its Fab fragment were tested with each biosensor to generate the association and dissociation rate constants, equilibrium association constants, and Gibbs free energy values for characterizing the binding event.

Animals↗