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author:("Tan, linzhi")
1.  X-ray structure of the SH3 domain of the phosphoinositide 3-kinase p85β subunit 
The crystal structure of the SH3 domain of the p85β subunit of human PI3K was determined to 2.0 Å resolution by molecular replacement. The overall structure is very similar to that of the p85α subunit of PI3K. The binding of two proline-rich ligand peptides to p85β SH3 was also characterized.
Src-homology 3 (SH3) domains are involved in extensive protein–protein interactions and constitute key elements of intracellular signal transduction. Three-dimensional structures have been reported for SH3 domains of various proteins, including the 85 kDa regulatory subunit (p85) of phosphoinositide 3-­kinase. However, all of the latter structures are of p85 isoform α and no crystal structure of the SH3 domain of the equally important isoform β has been reported to date. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.0 Å resolution of the SH3 domain of human p85β is described. The structure reveals a compact β-barrel fold very similar to that of p85α. However, binding studies with two classes of proline-rich ligand peptides demonstrate that the ligand-binding specificity differs slightly between the SH3 domains of human p85β and p85α, despite their high structural similarity.
PMCID: PMC3212445  PMID: 22102226
SH3 domains; p85β subunit; phosphoinositide 3-kinase
2.  3C Protease of Enterovirus 68: Structure-Based Design of Michael Acceptor Inhibitors and Their Broad-Spectrum Antiviral Effects against Picornaviruses 
Journal of Virology  2013;87(8):4339-4351.
We have determined the cleavage specificity and the crystal structure of the 3C protease of enterovirus 68 (EV68 3Cpro). The protease exhibits a typical chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional structure is closely related to that of the 3Cpro of rhinovirus 2, as well as to that of poliovirus. The phylogenetic position of the EV68 3Cpro between the corresponding enzymes of rhinoviruses on the one hand and classical enteroviruses on the other prompted us to use the crystal structure for the design of irreversible inhibitors, with the goal of discovering broad-spectrum antiviral compounds. We synthesized a series of peptidic α,β-unsaturated ethyl esters of increasing length and for each inhibitor candidate, we determined a crystal structure of its complex with the EV68 3Cpro, which served as the basis for the next design round. To exhibit inhibitory activity, compounds must span at least P3 to P1′; the most potent inhibitors comprise P4 to P1′. Inhibitory activities were found against the purified 3C protease of EV68, as well as with replicons for poliovirus and EV71 (50% effective concentration [EC50] = 0.5 μM for the best compound). Antiviral activities were determined using cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus serotypes. The most potent inhibitor, SG85, exhibited activity with EC50s of ≈180 nM against EV71 and ≈60 nM against human rhinovirus 14 in a live virus–cell-based assay. Even the shorter SG75, spanning only P3 to P1′, displayed significant activity (EC50 = 2 to 5 μM) against various rhinoviruses.
PMCID: PMC3624371  PMID: 23388726
3.  Structure-based identification of small molecule compounds targeting cell cyclophilin A with anti-HIV-1 activity 
European journal of pharmacology  2007;565(1-3):54-59.
Cyclophilin A acts as protein folding chaperones and intracellular transports in many cellular processes. Previous studies have shown that cyclophilin A can interact with HIV-1 (human immunodeficiency virus type 1) gag protein and enhance viral infectivity. Many cyclophilin A inhibitors such as cyclosporin A can inhibit HIV-1 replication in vitro. Here, we report a structure-based identification of novel non-peptidic cyclophilin A inhibitors as anti-HIV lead compounds. Following a computer-aided virtual screening and subsequent surface plasmon resonance (SPR) analysis, 12 low molecular weight cyclophilin A ligands were selected for further evaluation of their in vitro inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of cyclophilin A and HIV-1 replication. Five of these compounds (FD5, FD8, FD9, FD10 and FD12) exhibited inhibition against both PPIase activity and HIV-1 infection. These active compounds will be used as leads for structure and activity relationship (SAR) and optimization studies in order to design more effective anti-HIV-1 therapeutics, and as probes for investigating the effect of cyclophilins on HIV-1 replication.
PMCID: PMC3033441  PMID: 17449029
Cyclophilin A; Cyclosporin A; Human immunodeficiency virus; Peptidyl prolyl cis-trans isomerase
4.  The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes 
PLoS Pathogens  2009;5(5):e1000428.
Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.
Author Summary
The genome of the SARS coronavirus codes for 16 non-structural proteins that are involved in replicating this huge RNA (approximately 29 kilobases). The roles of many of these in replication (and/or transcription) are unknown. We attempt to derive conclusions concerning the possible functions of these proteins from their three-dimensional structures, which we determine by X-ray crystallography. Non-structural protein 3 contains at least seven different functional modules within its 1922-amino-acid polypeptide chain. One of these is the so-called SARS-unique domain, a stretch of about 338 residues that is completely absent from any other coronavirus. It may thus be responsible for the extraordinarily high pathogenicity of the SARS coronavirus, compared to other viruses of this family. We describe here the three-dimensional structure of the SARS-unique domain and show that it consists of two modules with a known fold, the so-called macrodomain. Furthermore, we demonstrate that these domains bind unusual nucleic-acid structures formed by consecutive guanosine nucleotides, where four strands of nucleic acid are forming a superhelix (so-called G-quadruplexes). SUD may be involved in binding to viral or host-cell RNA bearing this peculiar structure and thereby regulate viral replication or fight the immune response of the infected host cell.
PMCID: PMC2674928  PMID: 19436709

Results 1-4 (4)