A structural view of the conserved domain of rice stress-responsive NAC1

The importance of NAC(named as NAM,ATAF1,2,and CUC2)proteins in plant development,transcription regulation and regulatory pathways involving proteinprotein interactions has been increasingly recognized.We report here the high resolution crystal structure of SNAC1(stress-responsive NAC)NAC domain at 2.5Å.Although the structure of the SNAC1 NAC domain shares a structural similarity with the reported structure of the ANAC NAC1 domain,some key features,especially relating to two loop regions which potentially take the responsibility for DNA-binding,distinguish the SNAC1 NAC domain from other reported NAC structures.Moreover,the dimerization of the SNAC1 NAC domain is demonstrated by both soluble and crystalline conditions,suggesting this dimeric state should be conserved in this type of NAC family.Additionally,we discuss the possible NAC-DNA binding model according to the structure and reported biological evidences.

Structural view of the regulatory subunit of aspartate kinase from Mycobacterium tuberculosis

The aspartate kinase(AK)from Mycobacterium tuberculosis(Mtb)catalyzes the biosynthesis of aspartate family amino acids,including lysine,threonine,isoleucine and methionine.We determined the crystal structures of the regulatory subunit of aspartate kinase from Mtb alone(referred to as MtbAKβ)and in complex with threonine(referred to as MtbAKβ-Thr)at resolutions of 2.6A and 2.0Å,respectively.MtbAKβ is composed of two perpendicular non-equivalent ACT domains[aspartate kinase,chorismate mutase,and TyrA(prephenate dehydrogenase)]per monomer.Each ACT domain contains two α helices and four antiparallel β strands.The structure of MtbAKβ shares high similarity with the regulatory subunit of the aspartate kinase from Corynebacterium glutamicum(referred to as CgAKβ),suggesting similar regulatory mechanisms.Biochemical assays in our study showed that MtbAK is inhibited by threonine.Based on crystal structure analysis,we discuss the regulatory mechanism of MtbAK.

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

Enterovirus 71(EV71),one of the major causative agents for hand-foot-and-mouth disease(HFMD),has caused more than 100 deaths among Chinese children since March 2008.The EV71 genome encodes an RNAdependent RNA polymerase(RdRp),denoted 3D^(pol),which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics.Here we report the crystal structures of EV71 RdRp(3D^(pol))and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4Åresolution.The structure of EV71 RdRp(3D^(pol))has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp.And the EV71 RdRp(3D^(pol))complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding.The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus(FMDV)3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase.These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.

The newly emerged SARS-Like coronavirus HCoV-EMC also has an “Achilles’ heel”: current effective inhibitor targeting a 3C-like protease

From the global outbreak of SARS-CoV caused infection disease in 2003,coronaviruses(CoVs)are known to be a great threat to the human health.Recently,a new SARS-like coronavirus,human betacoronavirus 2c EMC/2012(HCoV-EMC),has been identified and the appearance of this new CoV raises concerns that a new spread of CoV may occurs in the future.By solving the crystal structure of HCoV-EMC main protease with a wide-spectrum anti-CoV inhibitor N3,we confi rmed that that N3 blocks the function of HCoV-EMC main protease through a similar mechanism to other CoVs.Together with the good pharmaceutical features,N3 is conceivable to be effective to HCoV-EMC and other CoVs appearing in the future.These fi ndings make it convincing that CoVs will not be a threat to human health.

Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses.The main protease(M^(pro))of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing.M^(pro) is initially produced as part of viral polyproteins and it is matured by autocleavage.Here,we report that,with the addition of an N-terminal extension peptide,M^(pro) can form a domain-swapped dimer.After complete removal of the extension peptide from the dimer,the mature M^(pro) self-assembles into a novel super-active octamer(AO-M^(pro)).The crystal structure of AO-M^(pro) adopts a novel fold with four domainswapped dimers packing into four active units with nearly identical conformation to that of the previously reported M^(pro) active dimer,and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains.Compared with the previously well characterized form of M^(pro),in equilibrium between inactive monomer and active dimer,the stable AO-M^(pro) exhibits much higher proteolytic activity at low concentration.As all eight active sites are bound with inhibitors,the polyvalent nature of the interaction between AO-M^(pro) and its polyprotein substrates with multiple cleavage sites,would make AO-M^(pro) functionally much more superior than the M^(pro) active dimer for polyprotein processing.Thus,during the initial period of SARS-CoV infection,this novel active form AOM^(pro) should play a major role in cleaving polyproteins as the protein level is extremely low.The discovery of AOM^(pro) provides new insights about the functional mechanism of M^(pro) and its maturation process.

New nsp8 isoform suggests mechanism for tuning viral RNA synthesis

During severe acute respiratory syndrome coronavirus(SARS-CoV)infection,the activity of the replication/transcription complexes(RTC)quickly peaks at 6 hours post infection(h.p.i)and then diminishes significantly in the late post-infection stages.This“down-up-down”regulation of RNA synthesis distinguishes different viral stages:primary translation,genome replication,and finally viron assembly.Regarding the nsp8 as the primase in RNA synthesis,we confirmed that the proteolysis product of the primase(nsp8)contains the globular domain(nsp8C),and indentified the resectioning site that is notably conserved in all the three groups of coronavirus.We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7,and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex.This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

Nascent polypeptide associated complex(NAC)and its two isolated subunits,αNAC and βNAC,play important roles in nascent peptide targeting.We determined a 1.9Åresolution crystal structure of the interaction core of NAC heterodimer and a 2.4Åresolution crystal structure ofαNAC NAC domain homodimer.These structures provide detailed information of NAC heterodimerization and αNAC homodimerization.We found that the NAC domains of αNAC and βNAC share very similar folding despite of their relative low identity of amino acid sequences.Furthermore,different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer.In addition,we successfully built a βNAC NAC domain homodimer model based on homologous modeling,suggesting that NAC domain dimerization is a general property of the NAC family.These 3D structures allow further studies on structurefunction relationship of NAC.

Crystallographic Studies of Cephalosporin Acylase from Pseudomonas sp. Strain 130

The cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C and/or glutaryl 7 aminocephalosporanic acid to produce 7 aminocephalosporanic acid. The cephalosporin acylase from Pseudomonas sp. strain 130 was crystallized in two different forms suitable for structural studies. A tetragonal crystal form diffracted to 0.24 nm belonged to the space group P4 12 12. There was one αβ heterodimer per asymmetric unit. A second crystal form diffracted to 0.21 nm belonged to the space group P2 1. There was four αβ heterodimers per asymmetric unit. The tetragonal crystal structure of CA 130 was determined using the multiwavelength anomalous diffraction method and the P2 1 crystal structure was then determined using the molecular replacement method.

Crystal structure of thermostable catechol 2,3-dioxygenase determined by multiwavelength anomalous dispersion method

The selenomethionyl derivative of the thermo-stable catechol 2,3-dioxygenase (SeMet-TC23O) is expressed, purified and crystallized. By using multiwave length anoma-lous dispersion (MAD) phasing techniques, the crystal structure of TC23O at 0.3 nm resolutions is determined. TC23O is a homotetramer. Each monomer is composed of N-terminal and C-terminal domains (residues 1-153 and 153-319, respectively). The two domains are proximately symmetric by a non-crystallographic axis. Each domain contains two characteristic motifs which are found in almost all of extradial dioxygenases.

Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6

Lysophosphatidic acid(LPA)is an important bioac-tive phospholipid involved in cell signaling through G-protein-coupled receptors pathways.It is also involved in balancing the lipid composition inside the cell,and modulates the function of lipid rafts as an intermediate in phospholipid metabolism.Because of its involvement in these important processes,LPA degradation needs to be regulated as precisely as its production.Lysophospha-tidic acid phosphatase type 6(ACP6)is an LPA-specifi c acid phosphatase that hydrolyzes LPA to monoacylglyc-erol(MAG)and phosphate.Here,we report three crystal structures of human ACP6 in complex with malonate,L-(+)-tartrate and tris,respectively.Our analyses revealed that ACP6 possesses a highly conserved Rossmann-fold-like body domain as well as a less conserved cap domain.The vast hydrophobic substrate-binding pocket,which is located between those two domains,is suitable for ac-commodating LPA,and its shape is different from that of other histidine acid phosphatases,a fact that is consistent with the observed difference in substrate preferences.Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis.The structure also indicates a water-supplying channel for substrate hydrolysis.Our structural data are consistent with the fact that the enzyme is active as a monomer.In combination with additional mutagenesis and enzyme activity studies,our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.