Biomedical subjects

Yihong Chen

Publications and source records attributed to Yihong Chen.

3 recordsLinked to original sources

PubMed2006

Structure, dynamics, and stability variation in bacterial albumin binding modules: implications for species specificity.

Protein G-related albumin-binding (GA) modules are frequently expressed on the surfaces of bacterial cells. The limited amino acid sequence variation among GA modules results in structural and functional differences with possible implications for bacterial pathogenesis and host specificity. In particular, the streptococcal G148-GA3 and F. magna ALB8-GA albumin-binding domains exhibit a degree of structural and dynamic diversity that may account for their varied affinities for different species of albumin. To explore the impact of GA module polymorphisms on albumin binding and specificity, we recently used offset recombinant PCR to shuffle seven artificially constructed representatives of the GA sequence space and scan the phage-displayed recombinant domains for mutations that supported binding to the phylogenetically distinct human and guinea pig serum albumins (HSA and GPSA) (Rozak et al. (2006) Biochemistry 45, 3263-3271). Surprisingly, phage selection revealed an overwhelming preference for a single recombinant domain (PSD-1, phage-selected domain-1) regardless of whether the phages were enriched for their abilities to bind one or both of these albumins. We describe here the NMR-derived structure, dynamics, and stability of unbound PSD-1. Our results demonstrate that increased flexibility is not a requirement for broadened specificity, as had been suggested earlier (Johansson et al. (2002) J. Mol. Biol. 316, 1083-1099), because PSD-1 binds the phylogenetically diverse HSA and GPSA even more tightly than G148-GA3 but is less flexible. The structural basis for albumin-binding specificity is analyzed in light of these new results.

Albumins↗

PubMed2006

Using offset recombinant polymerase chain reaction to identify functional determinants in a common family of bacterial albumin binding domains.

The 46 amino acid GA albumin binding module is a putative virulence factor that has been identified in 16 domains from four bacterial species. Aside from their possible effects on pathogenicity and host specificity, the natural genotypic and phenotypic variations that exist among members of this module offer unique opportunities for researchers to identify and explore functional determinants within the well-defined sequence space. We used a recently developed in vitro recombination technique, known as offset recombinant PCR, to shuffle seven homologues that encode a broad range of natural GA polymorphisms. Phage display and selection were applied to probe the recombinant library for members that showed simultaneous improvements to human and guinea pig serum albumin binding. Thermodynamic data for the most common phage-selected mutant suggest that domain-stabilizing mutations substantially improved GA binding for both species of albumin.

Albumins↗

PubMed2004

Specific recognition of the C-rich strand of human telomeric DNA and the RNA template of human telomerase by the first KH domain of human poly(C)-binding protein-2.

Poly(C)-binding proteins (PCBPs) constitute a family of nucleic acid-binding proteins that play important roles in a wide spectrum of regulatory mechanisms. The diverse functions of PCBPs are dependent on the ability of the PCBPs to recognize poly(C) sequences with high affinity and specificity. PCBPs contain three copies of KH (hnRNP K homology) domains, which are responsible for binding nucleic acids. We have determined the NMR structure of the first KH domain (KH1) from PCBP2. The PCBP2 KH1 domain adopts a structure with three alpha-helices packed against one side of a three-stranded antiparallel beta-sheet. Specific binding of PCBP2 KH1 to a number of poly(C) RNA and DNA sequences, including the C-rich strand of the human telomeric DNA repeat, the RNA template region of human telomerase, and regulatory recognition motifs in the poliovirus-1 5'-untranslated region, was established by monitoring chemical shift changes in protein (15)N-HSQC spectra. The nucleic acid binding groove was further mapped by chemical shift perturbation upon binding to a six-nucleotide human telomeric DNA. The binding groove is an alpha/beta platform formed by the juxtaposition of two alpha-helices, one beta-strand, and two flanking loops. Whereas there is a groove in common with all of the DNA and RNA binders with a hydrophobic floor accommodating a three-residue stretch of C residues, nuances in recognizing flanking residues are provided by hydrogen bonding partners in the KH domain. Specific interactions of PCBP2 KH1 with telomeric DNA and telomerase RNA suggest that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

Amino Acid Sequence↗