Biomedical subjects

Ann H Y Kwan

Publications and source records attributed to Ann H Y Kwan.

7 recordsLinked to original sources

PubMed2005

Zinc fingers as protein recognition motifs: structural basis for the GATA-1/friend of GATA interaction.

GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.

Binding Sites↗

PubMed2004

Crystal and solution structures of a superantigen from Yersinia pseudotuberculosis reveal a jelly-roll fold.

Superantigens are a class of microbial proteins with the ability to excessively activate T cells by binding to the T cell receptor. The staphylococcal and streptococcal superantigens are closely related in structure and possess an N-terminal domain that resembles an OB fold and a C-terminal domain similar to a beta-grasp fold. Yersinia pseudotuberculosis produces superantigens, YPMa, YPMb, and YPMc, which have no significant amino acid similarity to other proteins. We have determined the crystal and solution structures of YPMa, which show that the protein has a jelly-roll fold. The closest structural neighbors to YPMa are viral capsid proteins and members of the tumor necrosis factor superfamily. In the crystal structure, YPMa packs as a trimer, another feature shared with viral capsid proteins and TNF superfamily proteins. However, in solution YPMa behaves as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established.

Amino Acid Sequence↗

PubMed2003

Pentaprobe: a comprehensive sequence for the one-step detection of DNA-binding activities.

The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence-specific DNA-binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 4(5) possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 4(5) x 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double-stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA-binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N- and C-terminal fingers of GATA-1. We also showed that the N-terminal zinc finger domain of FOG-1 does not behave as a typical DNA-binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.

Algorithms↗

PubMed2003

Structural basis for the recognition of ldb1 by the N-terminal LIM domains of LMO2 and LMO4.

LMO2 and LMO4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T-cell leukemias. LMO4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. LMO2 and LMO4 interact with the ubiquitous nuclear adaptor protein ldb1/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldb1-LID). We report the solution structures of two LMO:ldb1 complexes (PDB: 1M3V and 1J2O) and show that ldb1-LID binds to the N-terminal LIM domain (LIM1) of LMO2 and LMO4 in an extended conformation, contributing a third strand to a beta-hairpin in LIM1 domains. These findings constitute the first molecular definition of LIM-mediated protein-protein interactions and suggest a mechanism by which ldb1 can bind a variety of LIM domains that share low sequence homology.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The structure of the zinc finger domain from human splicing factor ZNF265 fold.

Identification of the protein domains that are responsible for RNA recognition has lagged behind the characterization of protein-DNA interactions. However, it is now becoming clear that a range of structural motifs bind to RNA and their structures and molecular mechanisms of action are beginning to be elucidated. In this report, we have expressed and purified one of the two putative RNA-binding domains from ZNF265, a protein that has been shown to bind to the spliceosomal components U1-70K and U2AF35 and to direct alternative splicing. We show that this domain, which contains four highly conserved cysteine residues, forms a stable, monomeric structure upon the addition of 1 molar eq of Zn(II). Determination of the solution structure of this domain reveals a conformation comprising two stacked beta-hairpins oriented at approximately 80 degrees to each other and sandwiching the zinc ion; the fold resembles the zinc ribbon class of zinc-binding domains, although with one less beta-strand than most members of the class. Analysis of the structure reveals a striking resemblance to known RNA-binding motifs in terms of the distribution of key surface residues responsible for making RNA contacts, despite a complete lack of structural homology. Furthermore, we have used an RNA gel shift assay to demonstrate that a single crossed finger domain from ZNF265 is capable of binding to an RNA message. Taken together, these results define a new RNA-binding motif and should provide insight into the functions of the >100 uncharacterized proteins in the sequence data bases that contain this domain.

Amino Acid Sequence↗

PubMed2003

Engineering a protein scaffold from a PHD finger.

The design of proteins with tailored functions remains a relatively elusive goal. Small size, a well-defined structure, and the ability to maintain structural integrity despite multiple mutations are all desirable properties for such designer proteins. Many zinc binding domains fit this description. We determined the structure of a PHD finger from the transcriptional cofactor Mi2beta and investigated the suitability of this domain as a scaffold for presenting selected binding functions. The two flexible loops in the structure were mutated extensively by either substitution or expansion, without affecting the overall fold of the domain. A binding site for the corepressor CtBP2 was also grafted onto the domain, creating a new PHD domain that can specifically bind CtBP2 both in vitro and in the context of a eukaryotic cell nucleus. These results represent a step toward designing new regulatory proteins for modulating aberrant gene expression in vivo.

Amino Acid Sequence↗

PubMed2002

A new zinc binding fold underlines the versatility of zinc binding modules in protein evolution.

Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.

Amino Acid Sequence↗