The laboratory of Dr. Zhang studies structural and functional relations of macromolecules, mechanisms of protein folding and unfolding, The following are several areas that we are currently focusing on.
1. The mechanisms of protein folding and unfolding. Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of beta-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic inter¬actions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation. The characterization of the mechanism of protein folding is under progress.
2. Structural and functional characterization of sPLUNC1 protein. The short palate, lung and nasal epithelial clone 1 (SPLUNC1) protein is a member of the palate, lung, and nasal epithelium clone (PLUNC) family, also known as bactericidal/permeability-increasing (BPI) fold-containing protein, family A, member 1 (BPIFA1). SPLUNC1 is an abundant protein in human airways, but its function remains poorly understood. The lipid ligands of SPLUNC1 as well as other PLUNC family members are largely unknown, although some reports provide evidence that lipopolysaccharide (LPS) could be a lipid ligand. Unlike previous hypotheses, we found significant structural differences between SPLUNC1 and BPI. Recombinant SPLUNC1 produced in HEK 293 cells harbored several molecular species of sphingomyelin and phosphatidylcholine as its ligands. Significantly, in vitro lipid-binding studies failed to demonstrate interactions between SPLUNC1 and LPS, lipoteichoic acid, or polymyxin B. Instead, one of the major and most important pulmonary surfactant phospholipids, dipalmitoylphosphatidylcholine (DPPC), bound to SPLUNC1 with high affinity and specificity. We found that SPLUNC1 could be the first protein receptor for DPPC. These discoveries provide insight into the specific determinants governing the interaction between SPLUNC1 and lipids and also shed light on novel functions that SPLUNC1 and other PLUNC family members perform in host defense.
3. Single strand RNA demethylase JMJD6. JMJD6 binds alpha-ketoglutarate and iron and has been characterized as either a histone arginine demethylase or U2AF65 lysyl hydroxylase. However, we revealed a novel substrate binding groove and two positively charged surfaces. The structures also contain a stack of aromatic residues located near the active center. The side chain of one residue within this stack assumed different conformations in the two structures. Most interestingly, JMJD6 bound efficiently to single-stranded RNA, but not to single-stranded DNA, double-stranded RNA, or double-stranded DNA, or any protein. We concluded that JMJD6 is an ssRNA demethylase. To our delight, Dr. Michael Rosenfeld and Dr. Wei Liu’s groups found that JMJD6 specifically works on 5’ of 7SK RNA.
4. Structural and functional characterization of JmjC containing histone demethylases.
Covalent modifications of histone proteins, essential regulators of the activity of genes in eukaryotic cells, remodel the chromatin structure via a variety of enzymatic reactions. The reversible processes of some modifications, such as acetylation and deacetylation, are well characterized. Whether methylation and demethylation reversibly contribute to gene regulation, however, remains controversial. Recent studies have shown that methylation and demethylation are universally used to posttranslationally modify histones for the regulation of gene activity. In addition to LSD1, a nuclear amine oxidase homolog, which was found to function as a histone lysine demethylase, it was found that some JmjC domain-containing proteins are histone demethylases. Specifically, in collaboration with Dr. Yang Shi’s group at Harvard university, we found that members of the JMJD2 protein family are histone demethylases that act on trimethyl groups of H3-K9 and H3-K36. Moreover, some members of this family also have activity for dimethyl groups. To understand the relationships between the structures and functions of these proteins, we have determined the structure of the catalytic core of the JMJD2A protein. From this structure, several novel structure features were revealed, such as the novel JmjN domain, the JmjC domain, the C-terminal domain, and a zinc finger motif. These unique structural features create a potential catalytic center. The structure also revealed a characteristic signature motif, which includes structural determinants for cofactors such as Fe(II) and -ketoglutarate, a hallmark of non-heme containing oxygenases.
5. Characterization of Novel signal transduction mechanism through TALL-1 and its cognate receptors.
Trimer is the only functional unit for ligands of TNF family members, and trimer ligands recruit randomly distributed receptors on the membrane so as to trigger the down stream signal transduction. The novel virus-like structures of sTALL-1 with and without its cognate receptors determined in my lab are not consisted with the above common and well accepted theories. The sTALL-1 structure showed that sTALL-1 prefers existing at form of clustering with 20 trimers or 60 monomers through a novel “flap” region in vitro or vivo. Furthermore, the clustering state is the only functional unit. Structures of sTALL-1 with its cognate receptors, BCMA, BAFF-R, and TACI, showed that there are not only novel structural modules in these receptors (such as D2, X2, and others) but also novel interaction modes between ligand and receptors. These structure and interaction novelty underling novel signal transduction mechanism. Furthermore, structural modeling work on TALL-2/APRIL revealed a unique structural basis that distinglishes between sTALL-1 and APRIL for BAFF-R.
6. Structural and functional analysis of a new transcription family members.
Staphylococcus aureus (S. aureus) is a major pathogen in nosocomial bacteremia, generally accounting for ~15-20% of these bacteremic episodes. The expression of microbial virulence factors in S. aureus is a complex process that involves interactions among many gene products. Many of these virulence genes are regulated by global regulatory systems. One of them is the sar (staphylococcal accessory regulator) locus, which not only regulates the agr locus (accessory global regulator, containing protein or peptides, AgrA, AgrB, AgrC, and AgrD), but also directly controls the expression of several putative virulence determinants (e.g. hemolysins, fibrinogen and fibronectin binding proteins). A major transcription factor family plays pivotal roles in the regulation of the sar system, it is called SarA, We have determined several structures of fthe family members, SarR, SarA, and SarS etc. From these four structures, we further classified them in three sub families, SarA motif including SarA and SarR, which functions as homodime, SarS motif, which functions as monomer but contains two homolog SarA motif (as heterodimer), and RAT motif, which functions as homodimer, but contains additional helix bundle at its c-terminal.