Structural basis for DNA recognition and nuclease processing by the Mre11 homologue SbcD in double-strand breaks repair.

The Mre11 complex comprising meiotic recombination 11 (Mre11), Rad50 and Nijmegen breakage syndrome 1 (Nbs1) plays multiple important roles in the sensing, processing and repair of DNA double-strand breaks (DSBs). Here, crystal structures of the Escherichia coli Mre11 homologue SbcD and its Mn2+ complex are reported. Dimerization of SbcD depends on a four-helix bundle consisting of helices α2, α3, α2' and α3' of the two monomers, and the irregular and bent conformation of helices α3 and α3' in the SbcD dimer results in a dimeric arrangement that differs from those of previously reported Mre11 dimers. This finding indicates a distinct selectivity in DNA substrate recognition. The biochemical data combined with the crystal structures revealed that the SbcD monomer exhibits single-stranded DNA (ssDNA) endonuclease activity and double-stranded DNA (dsDNA) exonuclease activity on the addition of a high concentration of Mn2+. For the first time, atomic force microscopy analysis has been used to demonstrate that the SbcD monomer also possesses Mn2+-dependent dsDNA endonuclease activity. Loop ß7-α6 of SbcD is likely to be a molecular switch and plays an important role in the regulation of substrate binding, catalytic reaction and state transitions. Based on structural and mutational analyses, a novel ssDNA-binding model of SbcD is proposed, providing insight into the catalytic mechanism of DSBs repair by the Mre11 complex.

RecOR complex including RecR N-N dimer and RecO monomer displays a high affinity for ssDNA.

RecR is an important recombination mediator protein in the RecFOR pathway. RecR together with RecO and RecF facilitates RecA nucleoprotein filament formation and homologous pairing. Structural and biochemical studies of Thermoanaerobacter tengcongensis RecR (TTERecR) and its series mutants revealed that TTERecR uses the N-N dimer as a basic functional unit to interact with TTERecO monomer. Two TTERecR N-N dimers form a ring-shaped tetramer via an interaction between their C-terminal regions. The tetramer is a result of crystallization only. Hydrophobic interactions between the entire helix-hairpin-helix domains within the N-terminal regions of two TTERecR monomers are necessary for formation of a RecR functional N-N dimer. The TTERecR N-N dimer conformation also affects formation of a hydrophobic patch, which creates a binding site for TTERecO in the TTERecR Toprim domain. In addition, we demonstrate that TTERecR does not bind single-stranded DNA (ssDNA) and binds double-stranded DNA very weakly, whereas TTERecOR complex can stably bind DNA, with a higher affinity for ssDNA than double-stranded DNA. Based on these results, we propose an interaction model for the RecOR:ssDNA complex.

Growth phase-dependent reorganization of cryptophyte photosystem I antennae.

Photosynthetic cryptophytes are eukaryotic algae that utilize membrane-embedded chlorophyll a/c binding proteins (CACs) and lumen-localized phycobiliproteins (PBPs) as their light-harvesting antennae. Cryptophytes go through logarithmic and stationary growth phases, and may adjust their light-harvesting capability according to their particular growth state. How cryptophytes change the type/arrangement of the photosynthetic antenna proteins to regulate their light-harvesting remains unknown. Here we solve four structures of cryptophyte photosystem I (PSI) bound with CACs that show the rearrangement of CACs at different growth phases. We identify a cryptophyte-unique protein, PsaQ, which harbors two chlorophyll molecules. PsaQ specifically binds to the lumenal region of PSI during logarithmic growth phase and may assist the association of PBPs with photosystems and energy transfer from PBPs to photosystems.

Renoprotection by Inhibiting Connexin 43 Expression in a Mouse Model of Obesity-Related Renal Injury.

Introduction: Our previous study conducted in an obesity-related renal injury rat model have established a connection between increased connexin 43 (Cx43) expression and renal injury. In this study, we investigated whether inhibiting Cx43 expression could provide renoprotection in a mouse model of obesity-related renal injury. Methods: Five-week-old C57BL/6J mice were fed with a high-fat diet for 12 weeks to establish an obesity-related renal injury model, then they were treated with Cx43 antisense oligodeoxynucleotide (AS) or scrambled oligodeoxynucleotide (SCR) by an implanted osmotic pump for 4 weeks. Finally, the glomerular filtration function, the histological change in the glomeruli, and the markers of podocyte injury (WT-1, Nephrin) and inflammatory infiltration of renal tissue (CD68, F4/80 and VCAM-1) were examined respectively. Results: The results showed that inhibiting Cx43 expression by AS in this mouse model of obesity-related renal injury can effectively improve glomerular filtration function, alleviate glomerular expansion and podocyte injury, and attenuate the inflammatory infiltration of renal tissue. Conclusion: Our results demonstrated that inhibiting Cx43 expression by AS could provide renoprotection for the mouse model of obesity-related renal injury.

Crystal structures of SULT1A2 and SULT1A1 *3: insights into the substrate inhibition and the role of Tyr149 in SULT1A2.

The cytosolic sulfotransferases (SULTs) in vertebrates catalyze the sulfonation of endogenous thyroid/steroid hormones and catecholamine neurotransmitters, as well as a variety of xenobiotics, using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfonate donor. In this study, we determined the structures of SULT1A2 and an allozyme of SULT1A1, SULT1A1 *3, bound with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.4 and 2.3A resolution, respectively. The conformational differences between the two structures revealed a plastic substrate-binding pocket with two channels and a switch-like substrate selectivity residue Phe247, providing clearly a structural basis for the substrate inhibition. In SULT1A2, Tyr149 extends approximately 2.1A further to the inside of the substrate-binding pocket, compared with the corresponding His149 residue in SULT1A1 *3. Site-directed mutagenesis study showed that, compared with the wild-type SULT1A2, mutant Tyr149Phe SULT1A2 exhibited a 40 times higher K(m) and two times lower V(max) with p-nitrophenol as substrate. These latter data imply a significant role of Tyr149 in the catalytic mechanism of SULT1A2.

Crystal structure of spinach major light-harvesting complex at 2.72 A resolution.

The major light-harvesting complex of photosystem II (LHC-II) serves as the principal solar energy collector in the photosynthesis of green plants and presumably also functions in photoprotection under high-light conditions. Here we report the first X-ray structure of LHC-II in icosahedral proteoliposome assembly at atomic detail. One asymmetric unit of a large R32 unit cell contains ten LHC-II monomers. The 14 chlorophylls (Chl) in each monomer can be unambiguously distinguished as eight Chla and six Chlb molecules. Assignment of the orientation of the transition dipole moment of each chlorophyll has been achieved. All Chlb are located around the interface between adjacent monomers, and together with Chla they are the basis for efficient light harvesting. Four carotenoid-binding sites per monomer have been observed. The xanthophyll-cycle carotenoid at the monomer-monomer interface may be involved in the non-radiative dissipation of excessive energy, one of the photoprotective strategies that have evolved in plants.

Crystal structure of glycerophosphodiester phosphodiesterase (GDPD) from Thermoanaerobacter tengcongensis, a metal ion-dependent enzyme: insight into the catalytic mechanism.

Glycerophosphodiester phosphodiesterase (GDPD; EC 3.1.4.46) catalyzes the hydrolysis of a glycerophosphodiester to an alcohol and glycerol 3-phosphate in glycerol metabolism. It has an important role in the synthesis of a variety of products that participate in many biochemical pathways. We report the crystal structure of the Thermoanaerobacter tengcongensis GDPD (ttGDPD) at 1.91 A resolution, with a calcium ion and glycerol as a substrate mimic coordinated at this calcium ion (PDB entry 2pz0). The ttGDPD dimer with an intermolecular disulfide bridge and two hydrogen bonds is considered as the potential functional unit. We used site-directed mutagenesis to characterize ttGDPD as a metal ion-dependent enzyme, identified a cluster of residues involved in substrate binding and the catalytic reaction, and we propose a possible general acid-base catalytic mechanism for ttGDPD. Superposing the active site with the homologous structure GDPD from Agrobacterium tumefaciens (PDB entry 1zcc), which binds a sulfate ion in the active site, the sulfate ion can represent the phosphate moiety of the substrate, simulating the binding mode of the true substrate of GDPD.

Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis.

Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.

Crystal structure of earthworm fibrinolytic enzyme component B: a novel, glycosylated two-chained trypsin.

The earthworm fibrinolytic enzyme (EFE), belonging to a group of serine proteases with strong fibrinolytic activity, has been used in a mixture as an oral drug for prevention and treatment of thrombosis in East Asia. The EFE component b (EFE-b) is one of seven EFE components from Eisenia fetida, and among them it has nearly the highest fibrinolytic activity. Here, we report its crystal structure at a resolution of 2.06A. The structural analysis shows that EFE-b should be classified as a trypsin from earthworm. However, it is distinct from other trypsins. It is a two-chained protease with an N-terminal, pyroglutamated light chain and an N-glycosylated heavy chain. Furthermore, the heavy chain contains a novel structural motif, an eight-membered ring resulting from a disulfide bridge between two neighboring cysteine residues, and a cis peptide bond exists between these two cysteine residues. The crystal structure of EFE-b provides the structural basis for its high level of stability and reveals its complicated post-translational modifications in earthworm. This structure is the first reported for a glycosylated two-chained trypsin, which may provide useful clues to explain the origin and evolution of the chymotrypsin family.

Structure of HsdS subunit from Thermoanaerobacter tengcongensis sheds lights on mechanism of dynamic opening and closing of type I methyltransferase.

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase.

From structure to function: insights into the catalytic substrate specificity and thermostability displayed by Bacillus subtilis mannanase BCman.

BCman, a beta-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-beta-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the beta4 and beta7 strands, respectively. The 1.45-A crystal structure reveals that BCman is a typical (beta/alpha)(8) folding type. One large difference from the saddle-shaped active center of other endo-beta-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+1 and -1) within the active site of BCman. These two sites are involved in the enzyme's mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzyme's stability.

Crystal structure of human dynein light chain Dnlc2A: structural insights into the interaction with IC74.

The human light chain of the motor protein dynein, Dnlc2A, is also a novel TGF-beta-signaling component, which is altered with high frequency in epithelial ovarian cancer. It is an important mediator of dynein and the development of cancer, owing to its ability to bind to the dynein intermediate light chain (DIC) IC74 and to regulate TGF-beta-dependent transcriptional events. Here we report the 2.1-A crystal structure of Dnlc2A using single anomalous diffraction. The proteins form a homodimer in solution and interact mainly through the helix alpha(2), strand beta(3), and the loop following this strand in each protein to generate a 10-stranded beta-sheet core. The surface of the beta-sheet core is mainly positively charged and predicted (by software PPI-Pred) to be the site that interacts with other partners. At the same time, the residues 79-82, 88, and 90 of each molecule formed two holes in the core. Residue 89 of each molecule, which is crucial for the DIC binding function of Dnlc2A, is within the holes. On the basis of these observations, we propose that the homodimer is the structural and functional unit maintained by hydrogen bonding interactions and hydrophobic packing, and that the patch of the surface of the beta-sheet core is the main area of interaction with other partners. Furthermore, the two holes would be the key sites to interact with IC74.

1.42A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure.

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.

2.0 A crystal structure of human ARL5-GDP3'P, a novel member of the small GTP-binding proteins.

ARL5 is a member of ARLs, which is widespread in high eukaryotes and homologous between species. But no structure or biological function of this member is reported. We expressed, purified, and resolved the structure of human ARL5 with bound GDP3'P at 2.0 A resolution. A comparison with the known structures of ARFs shows that besides the typical features of ARFs, human ARL5 has specific features of its own. Bacterially expressed human ARL5 contains bound GDP3'P which is seldom seen in other structures. The hydrophobic tail of the introduced detergent Triton X-305 binds at the possible myristoylation site of Gly2, simulating the myristoylated state of N-terminal amphipathic helix in vivo. The structural features of the nucleotide binding motifs and the switch regions prove that ARL5 will undergo the typical GDP/GTP structural cycle as other members of ARLs, which is the basis of their biological functions.

Crystal structure of C-terminal desundecapeptide nitrite reductase from Achromobacter cycloclastes.

Monoclinic crystal structure of C-terminal desundecapeptide nitrite reductase (NiRc-11) from Achromobacter cycloclastes was determined at 2.6A. NiRc-11 exists as a loose trimer in the crystal. Deletion of 11 residues eliminates all intersubunit hydrogen bonds mediated by the C-terminal tail. The rigid irregular coil 105-112, which constitutes part of the sidewall of the active site pocket, undergoes conformational changes and becomes highly flexible in NiRc-11. Correspondingly, the linker segments between the two copper sites 95-100 and 135-136 are partly relaxed in conformation, which leads to disrupted active site microenvironments responsible for the activity loss and spectral change of NiRc-11. Comparison with the native structure revealed a bulky residue Met331 fastened by hydrogen bonding, which may play a direct role in keeping the right copper site geometry by protruding its side chain against the irregular coil 105-112. Sequence alignment showed that the bulky residue is conserved at position 331, indicating an equal importance of C-terminal segment in other copper-containing nitrite reductases.

Crystal structure of human sulfotransferase SULT1A3 in complex with dopamine and 3'-phosphoadenosine 5'-phosphate.

The human sulfotransferase, SULT1A3, catalyzes specifically the sulfonation of monoamines such as dopamine, epinephrine, and norepinephrine. SULT1A3 also has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is preferentially toward their D-form enantiomers and can be stimulated dramatically by Mn2+. To further our understanding of the molecular basis for the unique substrate specificity of this enzyme, we solved the crystal structure of human SULT1A3, complexed with dopamine and 3'-phosphoadenosine 5'-phosphate, at 2.6 A resolution and carried out autodocking analysis with D-Dopa. The structure of SULT1A3 enzyme-ligand complex clearly showed that residue Glu146 can form electrostatic interaction with dopamine and may play a pivotal role in the stereoselectivity and sulfating activity. On the other hand, residue Asp86 appeared to be critical to the Mn2+-stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3, in addition to a supporting role in the stereoselectivity and sulfating activity.

Structural basis for thermostability of beta-glycosidase from the thermophilic eubacterium Thermus nonproteolyticus HG102.

The three-dimensional structure of a thermostable beta-glycosidase (Gly(Tn)) from the thermophilic eubacterium Thermus nonproteolyticus HG102 was determined at a resolution of 2.4 A. The core of the structure adopts the (betaalpha)(8) barrel fold. The sequence alignments and the positions of the two Glu residues in the active center indicate that Gly(Tn) belongs to the glycosyl hydrolases of retaining family 1. We have analyzed the structural features of Gly(Tn) related to the thermostability and compared its structure with those of other mesophilic glycosidases from plants, eubacteria, and hyperthermophilic enzymes from archaea. Several possible features contributing to the thermostability of Gly(Tn) were elucidated.

Structural basis for broad substrate specificity of earthworm fibrinolytic enzyme component A.

Earthworm fibrinolytic enzyme component A (EFE-a) possesses an S1 pocket, which is typical for an elastase-like enzyme, but it can still hydrolyze varieties of substrates, and it exhibits wide substrate specificity. Former structure studies suggested that the four-residue insertion after Val(217) might endow EFE-a with this specificity. Based on the native crystal structure at a resolution of 2.3A, we improved the native crystal structure to 1.8A and determined its complex structure with the inhibitor Meo-Suc-Ala-Ala-Pro-Val-CMK at a resolution of 1.9A. The final structures show that: (1) EFE-a possesses multisubstrate-binding sites interacting with the substrates; (2) significant conformation adjustment takes place at two loops binding to the N-terminal of the substrates, which may enhance the interaction between the enzyme and the substrates. These characteristics make the substrate-specificity of EFE-a less dependent on the property of its S1-pocket and may endow the enzyme with the ability to hydrolyze chymotrypsin-specific substrates and even trypsin-specific substrates.

Crystal structure of KD93, a novel protein expressed in human hematopoietic stem/progenitor cells.

The crystal structure of a novel hypothetical protein, KD93, expressed in human hematopoietic stem/progenitor cells, was determined at 1.9A resolution using the multiple-wavelength anomalous dispersion (MAD) method. The protein KD93, which is encoded by the open reading frame HSPC031, is a NIP7 homologue and belongs to the UPF0113 family. The structural and functional information for the group of homologues has not yet been determined. Crystallographic analysis revealed that the overall fold of KD93 consists of two interlinked alpha/beta domains. Structure-based homology analysis with DALI revealed that the C domain of KD93 matches the PUA domain of some RNA modification enzymes, especially that of archaeosine tRNA-ribosyltransferase (ArcTGT), which suggests that its possible molecular function is related to RNA binding. The difference between the RNA binding regions of KD93 and ArcTGT in amino acid constitution and surface electrostatic potential indicate that they may have different RNA binding modes. The N domain of KD93 is a unique structure with no obvious similarity to other proteins with known three-dimensional structures. The high-resolution structure of KD93 provides a first view of a member of the family of hypothetical proteins. And the structure provides a framework to deduce and assay the molecular function of other proteins of the UPF0113 family.

pH-profile crystal structure studies of C-terminal despentapeptide nitrite reductase from Achromobacter cycloclastes.

Crystal structures of C-terminal despentapeptide nitrite reductase (NiRc-5) from Achromobacter cycloclastes were determined from 1.9 to 2.3A at pH 5.0, 5.4, and 6.2. NiRc-5, that has lost about 30% activity, is found to possess quite similar trimeric structures as the native enzyme. Electron density and copper content measurements indicate that the activity loss is not caused by the release of type 2 copper (T2Cu). pH-profile structural comparisons with native enzyme reveal that the T2Cu active center in NiRc-5 is perturbed, accounting for the partial loss of enzyme activity. This perturbation likely results from the less constrained conformations of two catalytic residues, Asp98 and His255. Hydrogen bonding analysis shows that the deletion of five residues causes a loss of more than half the intersubunit hydrogen bonds mediated by C-terminal tail. This study shows that the C-terminal tail plays an important role in controlling the conformations around the T2Cu site at the subunit interface, and helps keep the optimum microenvironment of active center for the full enzyme activity of AcNiR.