Biomedical subjects

Young-In Chi

Publications and source records attributed to Young-In Chi.

8 recordsLinked to original sources

PubMed2006

Crystallization of hepatocyte nuclear factor 1beta in complex with DNA.

Hepatocyte nuclear factor 1beta (HNF1beta) is a member of the POU transcription-factor family and binds the target DNA as a dimer with nanomolar affinity. The HNF1beta-DNA complex has been prepared and crystallized by hanging-drop vapor diffusion in 6%(v/v) PEG 300, 5%(w/v) PEG 8000, 8%(v/v) glycerol and 0.1 M Tris pH 8.0. The crystals diffracted to 3.2 A (93.9% completeness) using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group R3, with unit-cell parameters a = b = 172.69, c = 72.43 A. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure will shed light on the molecular mechanism of promoter recognition by HNF1beta and the molecular basis of the disease-causing mutations found in it.

Crystallization↗

PubMed2005

Homeodomain revisited: a lesson from disease-causing mutations.

The homeodomain is a highly conserved DNA-binding motif that is found in numerous transcription factors throughout a large variety of species from yeast to humans. These gene-specific transcription factors play critical roles in development and adult homeostasis, and therefore, any germline mutations associated with these proteins can lead to a number of congenital abnormalities. Although much has been revealed concerning the molecular architecture and the mechanism of homeodomain-DNA interactions, the study of disease-causing mutations can further provide us with instructive information as to the role of particular residues in a conserved mode of action. In this paper, I have compiled the homeodomain missense mutations found in various human diseases and re-examined the functional role of the mutational "hot spot" residues in light of the structures obtained from crystallography. These findings should be useful in understanding the essential components of the homeodomain and in attempts to design agonist or antagonists to modulate their activity and to reverse the effects caused by the mutations.

Amino Acid Sequence↗

PubMed2004

A phenylalanine zipper mediates APS dimerization.

The APS, SH2-B and LNK proteins are adapters that activate and modulate receptor tyrosine kinase and JAK/STAT signaling. We now show that a conserved N-terminal domain mediates APS homodimerization. We determined the crystal structure of the dimerization domain at a resolution of 1.7 A using bromide ion MAD phasing. Each molecule contributes two helices to a compact four-helix bundle having a bisecting-U topology. Its most conspicuous feature is a stack of interdigitated phenylalanine side chains at the domain core. These residues create a new motif we refer to as a 'phenylalanine zipper,' which is critical to dimerization. A newly developed bridging yeast tri-hybrid assay showed that APS dimerizes JAK2, insulin receptor and IGF1 receptor kinases using its SH2 and dimerization domains. Dimerization via the phenylalanine zipper domain provides a mechanism for activating and modulating tyrosine kinase activity even in the absence of extracellular ligands.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Structural basis for HNF-4alpha activation by ligand and coactivator binding.

In addition to suggesting that fatty acids are endogenous ligands, our recent crystal structure of HNF-4alpha showed an unusual degree of structural flexibility in the AF-2 domain (helix alpha12). Although every molecule contained a fatty acid within its ligand binding domain, one molecule in each homodimer was in an open inactive conformation with alpha12 fully extended and colinear with alpha10. By contrast, the second molecule in each homodimer was in a closed conformation with alpha12 folded against the body of the domain in what is widely considered to be the active state. This indicates that although ligand binding is necessary, it is not sufficient to induce an activating structural transition in HNF-4alpha as is commonly suggested to occur for nuclear receptors. To further assess potential mechanisms of activation, we have solved a structure of human HNF-4alpha bound to both fatty acid ligand and a coactivator sequence derived from SRC-1. The mode of coactivator binding is similar to that observed for other nuclear receptors, and in this case, all of the molecules adopt the closed active conformation. We conclude that for HNF-4alpha, coactivator rather than ligand binding locks the active conformation.

Binding Sites↗

PubMed2002

Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand.

HNF4 alpha is an orphan member of the nuclear receptor family with prominent functions in liver, gut, kidney and pancreatic beta cells. We have solved the x-ray crystal structure of the HNF4 alpha ligand binding domain, which adopts a canonical fold. Two conformational states are present within each homodimer: an open form with alpha helix 12 (alpha 12) extended and collinear with alpha 10 and a closed form with alpha 12 folded against the body of the domain. Although the protein was crystallized without added ligands, the ligand binding pockets of both closed and open forms contain fatty acids. The carboxylic acid headgroup of the fatty acid ion pairs with the guanidinium group of Arg(226) at one end of the ligand binding pocket, while the aliphatic chain fills a long, narrow channel that is lined with hydrophobic residues. These findings suggest that fatty acids are endogenous ligands for HNF4 alpha and establish a framework for understanding how HNF4 alpha activity is enhanced by ligand binding and diminished by MODY1 mutations.

Animals↗

PubMed2002

Zinc-mediated dimerization and its effect on activity and conformation of staphylococcal enterotoxin type C.

Staphylococcal enterotoxins are superantigen exotoxins that mediate food poisoning and toxic shock syndrome in humans. Despite their structural and functional similarities, superantigens display subtle differences in biological properties and modes of receptor binding as a result of zinc atoms bound differently in their crystal structures. For example, the crystal structures of the staphylococcal enterotoxins in the type C serogroup (SECs) contain a zinc atom coordinated by one aspartate and two histidine residues from one molecule and another aspartate residue from the next molecule, thus forming a dimer. This type of zinc ligation and zinc-mediated dimerization occurs in several SECs, but not in most other staphylococcal enterotoxin serogroups. This prompted us to investigate the potential importance of zinc in SEC-mediated pathogenesis. Site-directed mutagenesis was used to replace SEC zinc binding ligands with alanine. SEC mutants unable to bind zinc did not have major conformational alterations although they failed to form dimers. Zinc binding was not essential for T cell stimulation, emesis, or lethality although in general the mutants were less pyrogenic. Thus the zinc atom in SECs might represent a non-functional heavy atom in an exotoxin group that has diverged from related bacterial toxins containing crucial zinc atoms.

Animals↗

PubMed2002

Structure of the globular tail of nuclear lamin.

The nuclear lamins form a two-dimensional matrix that provides integrity to the cell nucleus and participates in nuclear activities. Mutations in the region of human LMNA encoding the carboxyl-terminal tail Lamin A/C are associated with forms of muscular dystrophy and familial partial lipodystrophy (FPLD). To help discriminate tissue-specific phenotypes, we have solved at 1.4-A resolution the three-dimensional crystal structure of the lamin A/C globular tail. The domain adopts a novel, all beta immunoglobulin-like fold. FPLD-associated mutations cluster within a small surface, whereas muscular dystrophy-associated mutations are distributed throughout the protein core and on its surface. These findings distinguish myopathy- and lipodystrophy-associated mutations and provide a structural framework for further testing hypotheses concerning lamin function.

Amino Acid Sequence↗

PubMed2002

Diabetes mutations delineate an atypical POU domain in HNF-1alpha.

Mutations in Hnf-1alpha are the most common Mendelian cause of diabetes mellitus. To elucidate the molecular function of a mutational hotspot, we cocrystallized human HNF-1alpha 83-279 with a high-affinity promoter and solved the structure of the complex. Two identical protein molecules are bound to the promoter. Each contains a homeodomain and a second domain structurally similar to POU-specific domains that was not predicted on the basis of amino acid sequence. Atypical elements in both domains create a stable interface that further distinguishes HNF-1alpha from other flexible POU-homeodomain proteins. The numerous diabetes-causing mutations in HNF-1alpha thus identified a previously unrecognized POU domain which was used as a search model to identify additional POU domain proteins in sequence databases.

Amino Acid Sequence↗