Cong-Zhao Zhou

Silk fibroin: Structural implications of a remarkable amino acid sequence

The amino acid sequence of the heavy chain of Bombyx mori silk fibroin was derived from the gene sequence. The 5,263‐residue (391‐kDa) polypeptide chain comprises 12 low‐complexity “crystalline” domains made up of Gly–X repeats and covering 94% of the sequence; X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats. The remainder includes a nonrepetitive 151‐residue header sequence, 11 nearly identical copies of a 43‐residue spacer sequence, and a 58‐residue C‐terminal sequence. The header sequence is homologous to the N‐terminal sequence of other fibroins with a completely different crystalline region. In Bombyx mori, each crystalline domain is made up of subdomains of ∼70 residues, which in most cases begin with repeats of the GAGAGS hexapeptide and terminate with the GAAS tetrapeptide. Within the subdomains, the Gly–X alternance is strict, which strongly supports the classic Pauling–Corey model, in which β‐sheets pack on each other in alternating layers of Gly/Gly and X/X contacts. When fitting the actual sequence to that model, we propose that each subdomain forms a β‐strand and each crystalline domain a two‐layered β‐sandwich, and we suggest that the β‐sheets may be parallel, rather than antiparallel, as has been assumed up to now. Proteins 2001;44:119–122.

Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate

2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena: the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms.

Activation of the LicT Transcriptional Antiterminator Involves a Domain Swing/Lock Mechanism Provoking Massive Structural Changes

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in β-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 Å resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ′ interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.

Crystal structure of the yeast phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 Å crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the β-sheet and α-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.

The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 Å resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 Å apart. The overall fold of API-A belongs to the β-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu87 and Lys145, and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met83 to P5′ Ala92, adopts a novel conformation with the Leu87 completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys141 to P5′ Glu150, binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.

Catalytic Mechanism and Structure of Viral Flavin-Dependent Thymidylate Synthase Thyx.

By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.

Refolding strategies from inclusion bodies in a structural genomics project

AbstractThe South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure (http://genomics.eu.org/). We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them. abbreviations: GdnHCl – guanidine hydrochloride; IPTG – isopropyl-β-d-thiogalactopyranoside; NMR – nuclear magnetic resonance spectroscopy; ORF – open reading frame; PCR – polymerase chain reaction; SDS-PAGE – sodium dodecylsulfate-polyacrylamide gel electrophoresis; TCA – trichloroacetic acid; β-SH – 2-mercaptoethanol.

Structural Insights into SraP-Mediated Staphylococcus aureus Adhesion to Host Cells

Staphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelets. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differ in calcium coordination pattern. Under the conditions tested, small-angle X-ray scattering and molecular dynamic simulation indicated that the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outwards. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated host-pathogen interaction of S. aureus.

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 A resolution revealed a novel two-strand antiparallel beta-sheet opposite the GSH binding groove. This extra beta-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.

Glutathionylation‐triggered conformational changes of glutaredoxin Grx1 from the yeast Saccharomyces cerevisiae

Glutaredoxins are thermo-stable thiol-disulfide oxidoreductases with different cellular functions, especially important in the cellular redox homeostasis. They are first identified as electron donors for ribonucleotide reductases,1 and also involved in a series of essential biosynthetic reactions and regulation in many biological processes as a glutathione-dependent manner.1–3 Glutaredoxins, coupled with NADPH, GSH, and glutathione reductase, consist of the glutaredoxin system, which transfers electrons from NADPH to glutaredoxins via GSH,4 protecting cells from damages caused by reactive oxygen species (ROS) during the course of normal aerobic metabolism or following exposure to radical-generating compounds, especially when the cellular survival mechanisms are unable to cope with the ROS and/or resulted damages.5,6 Structurally glutaredoxins (Grxs) belong to the Trx-like fold family, characterized with a b-pleated sheet at the center of the molecule surrounded by five helices and a dithiol active site CXXC motif, despite that some members in Grxs are monothiolic.3,7–11 Many studies have highlighted the key roles played by sulfhydryl groups ( SH) in response to oxidative stress, and particularly, the roles of the GSH/Grxs and thioredoxin (Trx) systems in maintaining the cellular redox homeostasis.12–15 To date five glutaredoxins (Grx1-5) have been identified in yeast S. cerevisiae and classified into two subfamilies. Grx1 and Grx2 belong to the dithiol glutaredoxin subfamily with the active site CPYC motif16 and Grx3-5 belong to the monothiol glutaredoxin subfamily with the active site CGFS motif.9 In dithiolic glutaredoxins the Nterminal cysteine commonly has a relatively low pKa and exists as thiolate in solution, which is important to thioldisulfide exchange reaction.17–20 The thiolate initializes nucleophilic attack on GSH-mixed disulfide to form a Grx-substrate intermediate, which can be reduced by the C-terminal cysteine, causing the formation of disulfide between two cysteines and the releasing of reduced substrate. Although a lot of glutaredoxin structures from E. coli, poxvirus, pig, and human have been reported, yeast Grx1 shares only 37% highest sequence identity with these structures. The crystal structure of GSH bound Grx1C30S mutant has been reported recently (PDB code 2JAC), however the distance between sulfur atoms of GSH and Cys27 is 2.88 Å, far beyond the normal disulfide bond length.21 The structure of glutathionylated Grx1 in our work has a standard length of disulfide bond and the side-chain orientation of Cys27 is different