Xiang Gao

The Arabidopsis Purple Acid Phosphatase AtPAP10 Is Predominantly Associated with the Root Surface and Plays an Important Role in Plant Tolerance to Phosphate Limitation

Induction of secreted acid phosphatase (APase) is a universal response of higher plants to phosphate (Pi) limitation. These enzymes are thought to scavenge Pi from organophosphate compounds in the rhizosphere and thus to increase Pi availability to plants when Pi is deficient. The tight association of secreted APase with the root surface may make plants more efficient in the utilization of soil Pi around root tissues, which is present in organophosphate forms. To date, however, no systematic molecular, biochemical, and functional studies have been reported for any of the Pi starvation-induced APases that are associated with the root surface after secretion. In this work, using genetic and molecular approaches, we identified Arabidopsis (Arabidopsis thaliana) Purple Acid Phosphatase10 (AtPAP10) as a Pi starvation-induced APase that is predominantly associated with the root surface. The AtPAP10 protein has phosphatase activity against a variety of substrates. Expression of AtPAP10 is specifically induced by Pi limitation at both transcriptional and posttranscriptional levels. Functional analyses of multiple atpap10 mutant alleles and overexpressing lines indicated that AtPAP10 plays an important role in plant tolerance to Pi limitation. Genetic manipulation of AtPAP10 expression may provide an effective means for engineering new crops with increased tolerance to Pi deprivation.

Cold Adaptation of Zinc Metalloproteases in the Thermolysin Family from Deep Sea and Arctic Sea Ice Bacteria Revealed by Catalytic and Structural Properties and Molecular Dynamics NEW INSIGHTS INTO RELATIONSHIP BETWEEN CONFORMATIONAL FLEXIBILITY AND HYDROGEN BONDING

Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, kcat/Km (10–40 °C), followed the order pseudolysin < MCP-02 < E495 with a ratio of ∼1:2:4. MCP-02 and E495 have the same optimal temperature (Topt, 57 °C, 5 °C lower than pseudolysin) and apparent melting temperature (Tm = 64 °C, ∼10 °C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new β-strand, nestling into two other β-strands. Simultaneously, the apparent Tm of the autoprocessed complex increases 20u2009°C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.

ROP11 GTPase Negatively Regulates ABA Signaling by Protecting ABI1 Phosphatase Activity from Inhibition by the ABA Receptor RCAR1/PYL9 in Arabidopsis

The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germination, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant-specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co-immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.

Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide

Significance DMS is an important participant in the global sulfur and carbon cycles. DMS oxidation products cause the formation of cloud condensation nuclei and hence may influence weather and climate. DMS is produced through the cleavage of dimethylsulfoniopropionate (DMSP) mainly by marine bacterial DMSP lyases. The molecular mechanism of DMSP cleavage to generate DMS remains unclear. In this study, the crystal structure of DddQ, a DMSP lyase, was solved, and detailed biochemical and structural analyses were performed. Our results also provided a foremost insight into the catalytic mechanism of the DMSP cleavage reaction. This study offers a better understanding of how marine bacteria cleave DMSP to generate the climatically important gas DMS. The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn2+ ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between “open” and “closed” states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction.

Molecular insights into the terminal energy acceptor in cyanobacterial phycobilisome

The linker protein LCM (ApcE) is postulated as the major component of the phycobilisome terminal energy acceptor (TEA) transferring excitation energy from the phycobilisome to photosystem II. LCM is the only phycobilin‐attached linker protein in the cyanobacterial phycobilisome through auto‐chromophorylation. However, the underlying mechanism for the auto‐chromophorylation of LCM and the detailed molecular architecture of TEA is still unclear. Here, we demonstrate that the N‐terminal phycobiliprotein‐like domain of LCM (Pfam00502, LP502) can specifically recognize phycocyanobilin (PCB) by itself. Biochemical assays indicated that PCB binds into the same pocket in LP502 as that in the allophycocyanin α‐subunit and that Ser152 and Asp155 play a vital role in LP502 auto‐chromophorylation. By carefully conducting computational simulations, we arrived at a rational model of the PCB‐LP502 complex structure that was supported by extensive mutational studies. In the PCB‐LP502 complex, PCB binds into a deep pocket of LP502 with a distorted conformation, and Ser152 and Asp155 form several hydrogen bonds to PCB fixing the PCB Ring A and Ring D. Finally, based on our results, the dipoles and dipole–dipole interactions in TEA are analysed and a molecular structure for TEA is proposed, which gives new insights into the energy transformation mechanism of cyanobacterial phycobilisome.

Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold.

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C‐N bonds, but DddP is deduced to cleave C‐S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensisu2005ITI_1157) bound to inhibitory 2‐(N‐morpholino) ethanesulfonic acid or PO43− and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion‐shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.

Elastolytic Mechanism of a Novel M23 Metalloprotease Pseudoalterin from Deep-sea Pseudoalteromonas sp. CF6-2 CLEAVING NOT ONLY GLYCYL BONDS IN THE HYDROPHOBIC REGIONS BUT ALSO PEPTIDE BONDS IN THE HYDROPHILIC REGIONS INVOLVED IN CROSS-LINKING

Background: The mechanism of marine elastin degradation is unclear. Results: A novel M23 metalloprotease pseudoalterin from a marine bacterium degraded elastin by cleaving both the glycyl bonds and the peptide bonds involved in cross-linking. Conclusion: Pseudoalterin adopts a novel elastolytic mechanism different from other M23 metalloproteases. Significance: The results shed light on the mechanism of marine elastin degradation. Elastin is a common insoluble protein that is abundant in marine vertebrates, and for this reason its degradation is important for the recycling of marine nitrogen. It is still unclear how marine elastin is degraded because of the limited study of marine elastases. Here, a novel protease belonging to the M23A subfamily, secreted by Pseudoalteromonas sp. CF6-2 from deep-sea sediment, was purified and characterized, and its elastolytic mechanism was studied. This protease, named pseudoalterin, has low identities (<40%) to the known M23 proteases. Pseudoalterin has a narrow specificity but high activity toward elastin. Analysis of the cleavage sites of pseudoalterin on elastin showed that pseudoalterin cleaves the glycyl bonds in hydrophobic regions and the peptide bonds Ala–Ala, Ala–Lys, and Lys–Ala involved in cross-linking. Two peptic derivatives of desmosine, desmosine-Ala-Ala and desmosine-Ala-Ala-Ala, were detected in the elastin hydrolysate, indicating that pseudoalterin can dissociate cross-linked elastin. These results reveal a new elastolytic mechanism of the M23 protease pseudoalterin, which is different from the reported mechanism where the M23 proteases only cleave glycyl bonds in elastin. Genome analysis suggests that M23 proteases may be popular in deep-sea sediments, implying their important role in elastin degradation. An elastin degradation model of pseudoalterin was proposed, based on these results and scanning electron microscopic analysis of the degradation by pseudoalterin of bovine elastin and cross-linked recombinant tropoelastin. Our results shed light on the mechanism of elastin degradation in deep-sea sediment.

Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family.

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP‐01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP‐01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate‐binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP‐01 displayed a non‐strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1’ site in collagen. His211 is a key residue for the P1‐basic‐residue preference of MCP‐01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.

Crystal structure of the N-terminal domain of linker L(R) and the assembly of cyanobacterial phycobilisome rods

Phycobilisomes are light‐harvesting supramolecular complexes in cyanobacteria and red algae. Linkers play a pivotal role in the assembly and energy transfer modulation of phycobilisomes. However, how linkers function remains unclear due to the lack of structural and biochemical studies of linkers, especially the N‐terminal domain of LR (pfam00427). Here, we report the crystal structure of the pfam00427 domain of the linker LR30 from Synechocystis sp. PCC 6803 at 1.9u2003Å. The pfam00427 presents as a previously uncharacterized point symmetric six α‐helix bundle. To elucidate the binding style of pfam00427 in the C‐phycocyanin (C‐PC) (αβ)6 hexamer, we fixed pfam00427 computationally into the C‐PC (αβ)6 inner cavity using the program AutoDock. Combined with a conserved ‘C‐PC binding patch’ on pfam00427 identified, we arrived at a model for the pfam00427–C‐PC (αβ)6 complex. This model was further optimized and evaluated as a reasonable result by a molecular dynamics simulation. In the resulting model, the pfam00427 domain is stably positioned in the central hole of the C‐PC trimer. Moreover, the LRT (pfam01383) was docked into our pfam00427–C‐PC model to generate a complete phycobilisome rod in which the linkers join individual biliprotein hexamers.