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1.  Role of the HIN Domain in Regulation of Innate Immune Responses 
The oligonucleotide/oligosaccharide binding (OB) fold is employed by proteins to bind nucleic acids during replication, transcription, and translation. Recently, a variation of the OB fold consisting of a tandem pair of OB folds named the HIN (hematopoietic expression, interferon-inducible nature, and nuclear localization) domain was shown to play essential roles in the regulation of innate immune responses originating from binding of nucleic acids in the cytoplasm or the nucleus of the cell. Although the two OB folds of the HIN domain are linked via a long linker region, conserved hydrophobic contacts between the two OB folds hold them together firmly, resulting in a single compact domain. This overall topology of the HIN domain seems to be highly conserved, and proteins containing the HIN domain have been grouped in the PYHIN family. Structures of the recently solved HIN domains reveal that these domains exhibit either absent in melanoma2 (Aim2) HIN-like or p202 HINa-like modes of DNA binding. These two modes of DNA binding seem to result in different responses and as a consequence confer distinct roles on the proteins. This review summarizes our current understanding of the structure and function of the HIN domains in context with the innate immune responses.
doi:10.1128/MCB.00857-13
PMCID: PMC3911281  PMID: 24164899
2.  Structure of Severe Fever with Thrombocytopenia Syndrome Virus Nucleocapsid Protein in Complex with Suramin Reveals Therapeutic Potential 
Journal of Virology  2013;87(12):6829-6839.
Severe fever with thrombocytopenia syndrome is an emerging infectious disease caused by a novel bunyavirus (SFTSV). Lack of vaccines and inadequate therapeutic treatments have made the spread of the virus a global concern. Viral nucleocapsid protein (N) is essential for its transcription and replication. Here, we present the crystal structures of N from SFTSV and its homologs from Buenaventura (BUE) and Granada (GRA) viruses. The structures reveal that phleboviral N folds into a compact core domain and an extended N-terminal arm that mediates oligomerization, such as tetramer, pentamer, and hexamer of N assemblies. Structural superimposition indicates that phleboviral N adopts a conserved architecture and uses a similar RNA encapsidation strategy as that of RVFV-N. The RNA binding cavity runs along the inner edge of the ring-like assembly. A triple mutant of SFTSV-N, R64D/K67D/K74D, almost lost its ability to bind RNA in vitro, is deficient in its ability to transcribe and replicate. Structural studies of the mutant reveal that both alterations in quaternary assembly and the charge distribution contribute to the loss of RNA binding. In the screening of inhibitors Suramin was identified to bind phleboviral N specifically. The complex crystal structure of SFTSV-N with Suramin was refined to a 2.30-Å resolution. Suramin was found sitting in the putative RNA binding cavity of SFTSV-N. The inhibitory effect of Suramin on SFTSV replication was confirmed in Vero cells. Therefore, a common Suramin-based therapeutic approach targeting SFTSV-N and its homologs could be developed for containing phleboviral outbreaks.
doi:10.1128/JVI.00672-13
PMCID: PMC3676114  PMID: 23576501
3.  Structural and Functional Analysis of STING Sheds New Light on Cyclic di-GMP Mediated Immune Signaling Mechanism 
Immunity  2012;36(6):10.1016/j.immuni.2012.03.019.
SUMMARY
STING is an essential signaling molecule for DNA and cyclic di-GMP (c-di-GMP)-mediated type I interferon (IFN) production via TANK-binding kinase 1 (TBK1) and Interferon regulatory factor 3 (IRF3) pathway. It contains an N-terminal transmembrane region and a cytosolic C-terminal domain (CTD). Here, we describe crystal structures of STING CTD alone and complexed with c-di-GMP in a unique binding mode. The strictly conserved AA153-173 region was shown to be cytosolic and participated in dimerization via hydrophobic interactions. The STING CTD functions as a dimer and the dimerization was independent of post-translational modifications. Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139-344) with TBK1. This suggests an extra TBK1 binding site, other than Ser358. This study provides a glimpse into the unique architecture of STING and sheds new light on the mechanism of c-di-GMP-mediated TBK1 signaling.
doi:10.1016/j.immuni.2012.03.019
PMCID: PMC3654694  PMID: 22579474
4.  A multi-dataset data-collection strategy produces better diffraction data 
Theoretical analysis and experimental validation prove that a multi-dataset data-collection strategy produces better diffraction data. The readiness test is a simple and sensitive method for X-ray data-collection system evaluation and a benchmark.
A multi-dataset (MDS) data-collection strategy is proposed and analyzed for macromolecular crystal diffraction data acquisition. The theoretical analysis indicated that the MDS strategy can reduce the standard deviation (background noise) of diffraction data compared with the commonly used single-dataset strategy for a fixed X-ray dose. In order to validate the hypothesis experimentally, a data-quality evaluation process, termed a readiness test of the X-ray data-collection system, was developed. The anomalous signals of sulfur atoms in zinc-free insulin crystals were used as the probe to differentiate the quality of data collected using different data-collection strategies. The data-collection results using home-laboratory-based rotating-anode X-ray and synchrotron X-ray systems indicate that the diffraction data collected with the MDS strategy contain more accurate anomalous signals from sulfur atoms than the data collected with a regular data-collection strategy. In addition, the MDS strategy offered more advantages with respect to radiation-damage-sensitive crystals and better usage of rotating-anode as well as synchrotron X-rays.
doi:10.1107/S0108767311037469
PMCID: PMC3211246  PMID: 22011470
multi-dataset data-collection strategy; readiness test
6.  Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented “EF-Hand-Like” Calcium-Binding Phage Lysin 
PLoS Pathogens  2014;10(5):e1004109.
The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.
Author Summary
The staphylococcal phage lysin LysGH15 demonstrates great potential against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the lytic activity of LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, we determined the structures of three individual LysGH15 domains using X-ray crystallography or nuclear magnetic resonance (NMR). The crystal structure unexpectedly reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove in the LysGH15 CHAP domain. Furthermore, the calcium ion plays an important role as a switch that modulates the lytic activity of the CHAP domain. Additionally, structure-guided mutagenesis also confirms that both E282 and the zinc ion play an important role in maintaining the lytic activity of the LysGH15 amidase-2 domain. Moreover, the NMR structure and titration-guided mutagenesis identify residues in the LysGH15 SH3b domain that are involved in the interactions with the substrate. The structure of LysGH15 is the first determined lysin structure from a staphylococcal phage, and these results represent a pivotal step forward in understanding this type of lysin.
doi:10.1371/journal.ppat.1004109
PMCID: PMC4022735  PMID: 24831957
7.  Mechanism of the Rpn13-induced activation of Uch37 
Protein & Cell  2014;5(8):616-630.
Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other’s ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37ΔHb,Hc,KEKE, a truncation removal of the C-terminal extension region (residues 256–329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270–407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-014-0046-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-014-0046-z
PMCID: PMC4130924  PMID: 24752541
Uch37-Rpn13 complex; de-ubiquitination; SAXS analysis; oligomerization; iso-peptidase
8.  Mechanism of the Rpn13-induced activation of Uch37 
Protein & Cell  2014;5(8):616-630.
Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other’s ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37ΔHb,Hc,KEKE, a truncation removal of the C-terminal extension region (residues 256–329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270–407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-014-0046-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-014-0046-z
PMCID: PMC4130924  PMID: 24752541
Uch37-Rpn13 complex; de-ubiquitination; SAXS analysis; oligomerization; iso-peptidase
9.  DDX41 recognizes bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response 
Nature immunology  2012;13(12):1155-1161.
Induction of type I interferons by the bacterial secondary messengers cyclic-di-GMP (c-di-GMP) or cyclic-di-AMP (c-di-AMP) is dependent on a signaling axis involving the STING adaptor, TBK1 kinase and IRF3 transcription factor. Here we identified the helicase DEAD box polypeptide 41 (DDX41) as a pattern recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via shRNA in murine or human cells inhibited the induction of innate immune genes and resulted in defective STING, TBK1 and IRF3 activation in response to c-di-GMP or c-di-AMP. These results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by the DDX41 PRR, which complexes with STING to signal to TBK1-IRF3 and activate the interferon response.
doi:10.1038/ni.2460
PMCID: PMC3501571  PMID: 23142775
10.  S-SAD phasing study of death receptor 6 and its solution conformation revealed by SAXS 
A comparative analysis of sulfur phasing of death receptor 6 (DR6) using data collected at wavelengths of 2.0 and 2.7 Å is presented. SAXS analysis of unliganded DR6 defines a dimer as the minimum physical unit in solution.
A subset of tumour necrosis factor receptor (TNFR) superfamily members contain death domains in their cytoplasmic tails. Death receptor 6 (DR6) is one such member and can trigger apoptosis upon the binding of a ligand by its cysteine-rich domains (CRDs). The crystal structure of the ectodomain (amino acids 1–348) of human death receptor 6 (DR6) encompassing the CRD region was phased using the anomalous signal from S atoms. In order to explore the feasibility of S-SAD phasing at longer wavelengths (beyond 2.5 Å), a comparative study was performed on data collected at wavelengths of 2.0 and 2.7 Å. In spite of sub-optimal experimental conditions, the 2.7 Å wavelength used for data collection showed potential for S-SAD phasing. The results showed that the R ano/R p.i.m. ratio is a good indicator for monitoring the anomalous data quality when the anomalous signal is relatively strong, while d′′/sig(d′′) calculated by SHELXC is a more sensitive and stable indicator applicable for grading a wider range of anomalous data qualities. The use of the ‘parameter-space screening method’ for S-SAD phasing resulted in solutions for data sets that failed during manual attempts. SAXS measurements on the ectodomain suggested that a dimer defines the minimal physical unit of an unliganded DR6 molecule in solution.
doi:10.1107/S0907444912004490
PMCID: PMC3335285  PMID: 22525750
sulfur phasing; SAXS analysis; long-wavelength X-rays; death receptor 6
11.  Structural View of a Non Pfam Singleton and Crystal Packing Analysis 
PLoS ONE  2012;7(2):e31673.
Background
Comparative genomic analysis has revealed that in each genome a large number of open reading frames have no homologues in other species. Such singleton genes have attracted the attention of biochemists and structural biologists as a potential untapped source of new folds. Cthe_2751 is a 15.8 kDa singleton from an anaerobic, hyperthermophile Clostridium thermocellum. To gain insights into the architecture of the protein and obtain clues about its function, we decided to solve the structure of Cthe_2751.
Results
The protein crystallized in 4 different space groups that diffracted X-rays to 2.37 Å (P3121), 2.17 Å (P212121), 3.01 Å (P4122), and 2.03 Å (C2221) resolution, respectively. Crystal packing analysis revealed that the 3-D packing of Cthe_2751 dimers in P4122 and C2221 is similar with only a rotational difference of 2.69° around the C axes. A new method developed to quantify the differences in packing of dimers in crystals from different space groups corroborated the findings of crystal packing analysis. Cthe_2751 is an all α-helical protein with a central hydrophobic core providing thermal stability via π:cation and π: π interactions. A ProFunc analysis retrieved a very low match with a splicing endonuclease, suggesting a role for the protein in the processing of nucleic acids.
Conclusions
Non-Pfam singleton Cthe_2751 folds into a known all α-helical fold. The structure has increased sequence coverage of non-Pfam proteins such that more protein sequences can be amenable to modelling. Our work on crystal packing analysis provides a new method to analyze dimers of the protein crystallized in different space groups. The utility of such an analysis can be expanded to oligomeric structures of other proteins, especially receptors and signaling molecules, many of which are known to function as oligomers.
doi:10.1371/journal.pone.0031673
PMCID: PMC3282739  PMID: 22363703
12.  Purification, crystallization and preliminary crystallographic analysis of the non-Pfam protein AF1514 from Archeoglobus fulgidus DSM 4304 
A non-Pfam protein, AF1514, from A. fulgidus has been crystallized.
A 10.5 kDa non-Pfam hypothetical protein, AF1514, from the hyperthermophilic archaeon Archeoglobus fulgidus has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 2.09 Å resolution and a data set was collected at 100 K using Cu Kα radiation from a rotating-anode X-ray source. The crystals belong to space group P41212 or P43212, with unit-cell parameters a = b = 49.27, c = 106.61 Å. The calculated Matthews coefficient was 3.16 Å3 Da−1, suggesting the presence of one molecule in the asymmetric unit.
doi:10.1107/S1744309107068649
PMCID: PMC2374175  PMID: 18259057
non-Pfam proteins; reductive methylation
13.  Structure of the minimized α/β-hydrolase fold protein from Thermus thermophilus HB8 
Acta Crystallographica Section F  2007;63(Pt 12):993-997.
The crystal structure of the minimized α/β-hydrolase fold protein encoded by the gene TTHA1544 from T. thermophilus HB8 has been determined at 2.0 Å resolution.
The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 Å resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four α-helices and six β-strands, with the β-strands composing a central β-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the α/β-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized α/β-hydrolase fold and that an additional component would be required for its activity.
doi:10.1107/S1744309107061106
PMCID: PMC2344104  PMID: 18084077
T. thermophilus HB8; TTHA1544; α/β-hydrolase fold; singleton
14.  Structural Basis and Catalytic Mechanism for the Dual Functional Endo-β-N-Acetylglucosaminidase A 
PLoS ONE  2009;4(3):e4658.
Endo-β-N-acetylglucosaminidases (ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have become the focus of intense research because of their potential for synthesis of glycopeptides. We have determined the 3D structures of an ENGase from Arthrobacter protophormiae (Endo-A) in 3 forms, one in native form, one in complex with Man3GlcNAc-thiazoline and another in complex with GlcNAc-Asn. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. The conserved essential catalytic residues – E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as “gate-keepers”. Interestingly, Y299F mutation resulted in a 3 fold increase in the transglycosylation activity. The structure provides insights into the catalytic mechanism of GH85 family of glycoside hydrolases at molecular level and could assist rational engineering of ENGases.
doi:10.1371/journal.pone.0004658
PMCID: PMC2646837  PMID: 19252736
15.  Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus  
The molybdenum-cofactor biosynthesis protein C from T. thermophilus has been crystallized in two different space groups, P21 and R32; the crystals diffracted to 1.9 and 1.75 Å resolution, respectively.
The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P21, with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 Å, β = 104.9°; the crystal diffracted to a resolution of 1.9 Å. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 Å, and diffracted to 1.75 Å resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P21 and R32, respectively.
doi:10.1107/S1744309106052560
PMCID: PMC2330104  PMID: 17183168
thermophilic microorganisms; Thermus thermophilus; molybdenum-cofactor biosynthesis; MoaC
16.  Structure of the hypothetical protein PF0899 from Pyrococcus furiosus at 1.85 Å resolution 
Acta Crystallographica Section F  2007;63(Pt 7):549-552.
The crystal structure of the hypothetical protein PF0899 from P. furiosus has been determined to 1.85 Å resolution.
The hypothetical protein PF0899 is a 95-residue peptide from the hyperthermophilic archaeon Pyrococcus furiosus that represents a gene family with six members. P. furiosus ORF PF0899 has been cloned, expressed and crystallized and its structure has been determined by the Southeast Collaboratory for Structural Genomics (http://www.secsg.org). The structure was solved using the SCA2Structure pipeline from multiple data sets and has been refined to 1.85 Å against the highest resolution data set collected (a presumed gold derivative), with a crystallographic R factor of 21.0% and R free of 24.0%. The refined structure shows some structural similarity to a wedge-shaped domain observed in the structure of the major capsid protein from bacteriophage HK97, suggesting that PF0899 may be a structural protein.
doi:10.1107/S1744309107024049
PMCID: PMC2335137  PMID: 17620707
structural genomics; SECSG; Pfu-871755; PF0899; high-throughput structure
17.  Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7 Å resolution 
Two acostatin heterodimers interact together to form an αββα tetramer.
Disintegrins are a family of small (4–14 kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an αβ heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon contortrix contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an R work and R free of 18.6% and 21.5%, respectively, using all data in the 20–1.7 Å resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-­terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short β-sheets, each of which is formed by a pair of antiparallel β-strands connected by β-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the αβ chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an αββα acostatin tetramer. Further experiments are required to identify whether the αββα acostatin complex plays a functional role in vivo.
doi:10.1107/S0907444908002370
PMCID: PMC2631110  PMID: 18391413
disintegrins; acostatin; tetramer
18.  The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus▿  
Journal of Bacteriology  2007;189(16):6057-6067.
We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the kcat/Km value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108°C that increases to 112°C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-Å resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.
doi:10.1128/JB.00151-07
PMCID: PMC1952034  PMID: 17545282
19.  (NZ)CH...O Contacts assist crystallization of a ParB-like nuclease 
Background
The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization
Results
Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ) CH...O contacts (3.2 – 3.7 Å) by the addition of 2 methyl groups to the side chain amine nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 Å resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization.
Conclusion
In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O contacts could provide a solution to crystallization problems for a broad range of protein targets.
doi:10.1186/1472-6807-7-46
PMCID: PMC1940005  PMID: 17617922
20.  Isolation, crystallization and preliminary X-ray analysis of a methanol-induced corrinoid protein from Moorella thermoacetica  
Strongly diffracting crystals of a methanol-induced corrinoid protein from M. thermoacetica have been obtained.
A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 Å resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 Å. N-­terminal amino-acid sequencing indicates that the crystals contain a C-­terminal fragment of the protein.
doi:10.1107/S1744309105010511
PMCID: PMC1952305  PMID: 16511090
corrinoid; methyltransferases

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