SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation.

An effective Severe Acute Respiratory Syndrome (SARS) vaccine is likely to include components that can induce specific cytotoxic T-lymphocyte (CTL) responses. The specificities of such responses are governed by human leukocyte antigen (HLA)-restricted presentation of SARS-derived peptide epitopes. Exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information (Lauemoller et al., Rev Immunogenet 2001: 2: 477-91). The latter was recently established when a causative coronavirus (SARS-CoV) was isolated and full-length sequenced (Marra et al., Science 2003: 300: 1399-404). Here, we have combined advanced bioinformatics and high-throughput immunology to perform an HLA supertype-, genome-wide scan for SARS-specific CTL epitopes. The scan includes all nine human HLA supertypes in total covering >99% of all individuals of all major human populations (Sette & Sidney, Immunogenetics 1999: 50: 201-12). For each HLA supertype, we have selected the 15 top candidates for test in biochemical binding assays. At this time (approximately 6 months after the genome was established), we have tested the majority of the HLA supertypes and identified almost 100 potential vaccine candidates. These should be further validated in SARS survivors and used for vaccine formulation. We suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design.

Prediction of protein secondary structure at 80% accuracy.

Secondary structure prediction involving up to 800 neural network predictions has been developed, by use of novel methods such as output expansion and a unique balloting procedure. An overall performance of 77.2%-80.2% (77.9%-80.6% mean per-chain) for three-state (helix, strand, coil) prediction was obtained when evaluated on a commonly used set of 126 protein chains. The method uses profiles made by position-specific scoring matrices as input, while at the output level it predicts on three consecutive residues simultaneously. The predictions arise from tenfold, cross validated training and testing of 1032 protein sequences, using a scheme with primary structure neural networks followed by structure filtering neural networks. With respect to blind prediction, this work is preliminary and awaits evaluation by CASP4.

Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site.

Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP). These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and which interacts with the ribose 5-phosphate moiety in structurally characterized complexes of PRPP and nucleotides. We show that each class of phosphoribosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) have a proline residue at a position where other phosphoribosyltransferases and the PRPP synthases have aspartate. To investigate the role of this unusual proline (Pro 131 in the E. coli UPRTase) for enzyme activity, we changed the residue to an aspartate and purified the mutant P131D enzyme to compare its catalytic properties with the properties of the wild-type protein. We found that UPRTase of E. coli obeyed the kinetics of a sequential mechanism with the binding of PRPP preceding the binding of uracil. The basic kinetic constants were derived from initial velocity measurements, product inhibition, and ligand binding assays. The change of Pro 131 to Asp caused a 50-60-fold reduction of the catalytic rate (kcat) in both directions of the reaction and approximately a 100-fold increase in the KM for uracil. The KM for PRPP was strongly diminished by the mutation, but kcat/KM,PRPP and the dissociation constant (KD,PRPP) were nearly unaffected. We conclude that the proline in the PRPP binding site of UPRTase is of only little importance for binding of PRPP to the free enzyme, but is critical for binding of uracil to the enzyme-PRPP complex and for the catalytic rate.

Kinetic mechanism of OMP synthase: a slow physical step following group transfer limits catalytic rate.

Orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10) forms the UMP precursor orotidine 5'-monophophate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). Here, equilibrium binding, isotope partitioning, and chemical quench studies were used to determine rate and equilibrium constants for the kinetic mechanism. PRPP bound to two sites per dimer with a KD of 33 microM. Binding of OMP and orotate also occurred to a single class of two sites per dimer, with KD values of 3 and 280 microM, respectively. Pyrophosphate binding to two sites was weak with a KD of 960 microM, and in the presence of bound orotate, its affinity for the first site was enhanced 4-fold (KD = 230 microM). Preformed E.OMP, E.PRPP, E.PPi, and E.orotate complexes were trapped as products in isotope partitioning experiments, indicating that each was catalytically competent and confirming a random mechanism. Rapid quench experiments revealed burst kinetics for product formation in both the forward phosphoribosyltransferase and the reverse pyrophosphorolysis reactions. The steady-state rate in the forward reaction was preceded by a burst (nfwd = 1.5/dimer) of at least 300 s-1. In the pyrophosphorolysis reaction, a burst (nrev = 0.7/dimer; k >/= 300 s-1) was also noted. These results allowed us to develop a complete kinetic mechanism for OPRTase, in which a rapid phosphoribosyl transfer reaction at equilibrium is followed by a slow step involving release of product. When the microviscosity, etarel, of the reaction medium was increased with sucrose, the forward kcat decreased in proportion to etarel with a slope of 0.8. In the reverse reaction a more limited dependence of kcat (slope = 0. 3) was observed. On the basis of the known structures of OPRTase, we propose that a highly conserved, catalytically important, solvent-exposed loop descends during catalysis to shield the active site. In the accompanying paper, the slow product release step is shown to relate to movement of the solvent-exposed loop.

The DNA damage-inducible dinD gene of Escherichia coli is equivalent to orfY upstream of pyrE.

The DNA damage-inducible gene dinD, originally identified by Kenyon and Walker (C. J. Kenyon and G. C. Walker, Proc. Natl. Acad. Sci. USA 77:2819-2823, 1980) by selection of the dinD::MudI (Ap lac) fusion, is shown here to be equivalent to the open reading frame orfY near pyrE. The evidence for identity between the two genes includes results from P1 transduction, Southern hybridization, and cloning and sequencing of the dinD fusion. No data were obtained that reveal any hints about the function of the dinD gene.