Crystal Structure of PhnH: an Essential Component of Carbon-Phosphorus Lyase in Escherichia coli

ABSTRACT Organophosphonates are reduced forms of phosphorous that are characterized by the presence of a stable carbon-phosphorus (C-P) bond, which resists chemical hydrolysis, thermal decomposition, and photolysis. The chemically inert nature of the C-P bond has raised environmental concerns as toxic phosphonates accumulate in a number of ecosystems. Carbon-phosphorous lyase (CP lyase) is a multienzyme pathway encoded by the phn operon in gram-negative bacteria. In Escherichia coli 14 cistrons comprise the operon (phnCDEFGHIJKLMNOP) and collectively allow the internalization and degradation of phosphonates. Here we report the X-ray crystal structure of the PhnH component at 1.77 Å resolution. The protein exhibits a novel fold, although local similarities with the pyridoxal 5′-phosphate-dependent transferase family of proteins are apparent. PhnH forms a dimer in solution and in the crystal structure, the interface of which is implicated in creating a potential ligand binding pocket. Our studies further suggest that PhnH may be capable of binding negatively charged cyclic compounds through interaction with strictly conserved residues. Finally, we show that PhnH is essential for C-P bond cleavage in the CP lyase pathway.

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Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798315024729 ◽

2016 ◽

Vol 72 (2) ◽

pp. 236-244 ◽

Cited By ~ 7

Author(s):

Zhen Chen ◽

Li-Hong Zhan ◽

Hai-Feng Hou ◽

Zeng-Qiang Gao ◽

Jian-Hua Xu ◽

...

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Outer Membrane ◽

Essential Role ◽

Biochemical Analysis ◽

Hydrophobic Interactions ◽

Outer Membrane Proteins ◽

Structural Basis ◽

Conserved Residues ◽

Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

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Crystal structure of TchmY from Actinoplanes teichomyceticus

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19010914 ◽

2019 ◽

Vol 75 (9) ◽

pp. 570-575

Author(s):

Zhenzhen Yang ◽

Lilan Zhang ◽

Xuejing Yu ◽

Shan Wu ◽

Yong Yang ◽

...

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Gene Clusters ◽

Binding Pocket ◽

Animal Nutrition ◽

Biosynthetic Pathways ◽

Substrate Binding Pocket ◽

Actinoplanes Teichomyceticus ◽

Unknown Sequence ◽

Potential Substrate

Moenomycin-type antibiotics are phosphoglycolipids that are notable for their unique modes of action and have proven to be useful in animal nutrition. The gene clusters tchm from Actinoplanes teichomyceticus and moe from Streptomyces are among a limited number of known moenomycin-biosynthetic pathways. Most genes in tchm have counterparts in the moe cluster, except for tchmy and tchmz, the functions of which remain unknown. Sequence analysis indicates that TchmY belongs to the isoprenoid enzyme C2-like superfamily and may serve as a prenylcyclase. The enzyme was proposed to be involved in terminal cyclization of the moenocinyl chain in teichomycin, leading to the diumycinol chain of moenomycin isomers. Here, recombinant TchmY protein was expressed in Escherichia coli and its crystal structure was solved by SIRAS. Structural analysis and comparison with other prenylcyclases were performed. The overall fold of TchmY consists of an (α/α)6-barrel, and a potential substrate-binding pocket is found in the central chamber. These results should provide important information regarding the biosynthetic basis of moenomycin antibiotics.

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Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423194112 ◽

2014 ◽

Vol 112 (1) ◽

pp. 142-147 ◽

Cited By ~ 3

Author(s):

Nimisha Singh ◽

Günter Blobel ◽

Hang Shi

Keyword(s):

Crystal Structure ◽

Binding Pocket ◽

Binding Activity ◽

Stable Complex ◽

Mrna Transport ◽

Conserved Residues ◽

Motor Complex ◽

Targeted Transport ◽

High Affinity Site

The segregation of approximately two dozen distinct mRNAs from yeast mother to daughter cell cytoplasm is a classical paradigm for eukaryotic mRNA transport. The information for transport resides in an mRNA element 40–100 nt in length, known as “zipcode.” Targeted transport requires properly positioned actin filaments and cooperative loading of mRNA cargo to myosin. Cargo loading to myosin uses myosin 4 protein (Myo4p), swi5p-dependent HO expression 2 protein (She2p) and 3 protein (She3p), and zipcode. We previously determined a crystal structure of Myo4p and She3p, their 1:2 stoichiometry and interactome; we furthermore showed that the motor complex assembly requires two Myo4p⋅She3p heterotrimers, one She2p tetramer, and at least a single zipcode to yield a stable complex of [Myo4p⋅She3p⋅She2p⋅zipcode] in 2:4:4:1 stoichiometry in vitro. Here, we report a structure at 2.8-Å resolution of a cocrystal of a She2p tetramer bound to a segment of She3p. In this crystal structure, the She3p segment forms a striking hook that binds to a shallow hydrophobic pocket on the surface of each She2p subunit of the tetramer. Both She3p hook and cognate She2p binding pocket are composed of highly conserved residues. We also discovered a highly conserved region of She3p upstream of its hook region. Because this region consists of basic and aromatic residues, it likely represents part of She3p’s binding activity for zipcode. Because She2p also exhibits zipcode-binding activity, we suggest that “hooking” She3p onto She2p aligns each of their zipcode-binding activities into a high-affinity site, thereby linking motor assembly to zipcode.

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Biochemical and structural insights into the activation of PBP1b by the essential domain of FtsN

10.1101/2020.06.05.136150 ◽

2020 ◽

Author(s):

Adrien Boes ◽

Frederic Kerff ◽

Raphael Herman ◽

Thierry Touze ◽

Eefjan Breukink ◽

...

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Cell Wall ◽

Cell Division ◽

Binding Pocket ◽

Nonpermissive Temperature ◽

Multiprotein Complex ◽

Division Site ◽

Structural Insights

AbstractPeptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division PG synthesis localizes at mid-cell under the control of a multiprotein complex, the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. The region L75 to Q93 of FtsN (EFtsN) was shown to be essential and sufficient for its functioning in vivo but the specific target and the molecular mechanism remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its GTase activity. We also report the crystal structure of PBP1b in complex with EFtsN which provides structural insights into the mode of binding of EFtsN at the junction between the GTase and UB2H domains of PBP1b. Interestingly, the mutations R141A/R397A of PBP1b, within the EFtsN binding pocket, reduce the activation of PBP1b by FtsN. This mutant was unable to rescue ΔponB-ponAts strain at nonpermissive temperature and induced a mild cell chaining phenotype and cell lysis. Altogether, the results show that PBP1b is a target of EFtsN and suggest that binding of FtsN to PBP1b contributes to trigger septal PG synthesis and cell constriction.

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Crystal structure of human LDB1 in complex with SSBP2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914181117 ◽

2019 ◽

Vol 117 (2) ◽

pp. 1042-1048 ◽

Cited By ~ 2

Author(s):

Hongyang Wang ◽

Juhyun Kim ◽

Zhizhi Wang ◽

Xiao-Xue Yan ◽

Ann Dean ◽

...

Keyword(s):

Crystal Structure ◽

Cell Fate ◽

Long Range ◽

Binding Proteins ◽

Binding Pocket ◽

Structural Basis ◽

Globin Genes ◽

Dimerization Domain ◽

Cell Fate Decisions ◽

Potential Ligand

The Lim domain binding proteins (LDB1 and LDB2 in human and Chip in Drosophila) play critical roles in cell fate decisions through partnership with multiple Lim-homeobox and Lim-only proteins in diverse developmental systems including cardiogenesis, neurogenesis, and hematopoiesis. In mammalian erythroid cells, LDB1 dimerization supports long-range connections between enhancers and genes involved in erythropoiesis, including the β-globin genes. Single-stranded DNA binding proteins (SSBPs) interact specifically with the LDB/Chip conserved domain (LCCD) of LDB proteins and stabilize LDBs by preventing their proteasomal degradation, thus promoting their functions in gene regulation. The structural basis for LDB1 self-interaction and interface with SSBPs is unclear. Here we report a crystal structure of the human LDB1/SSBP2 complex at 2.8-Å resolution. The LDB1 dimerization domain (DD) contains an N-terminal nuclear transport factor 2 (NTF2)-like subdomain and a small helix 4–helix 5 subdomain, which together form the LDB1 dimerization interface. The 2 LCCDs in the symmetric LDB1 dimer flank the core DDs, with each LCCD forming extensive interactions with an SSBP2 dimer. The conserved linker between LDB1 DD and LCCD covers a potential ligand-binding pocket of the LDB1 NTF2-like subdomain and may serve as a regulatory site for LDB1 structure and function. Our structural and biochemical data provide a much-anticipated structural basis for understanding how LDB1 and the LDB1/SSBP interactions form the structural core of diverse complexes mediating cell choice decisions and long-range enhancer–promoter interactions.

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The molecular structure of the μ‎-opioid receptor

10.1093/med/9780198834359.003.0038 ◽

2018 ◽

Author(s):

Roger D. Knaggs

Keyword(s):

Crystal Structure ◽

Side Effects ◽

Opioid Receptor ◽

Three Dimensional ◽

Binding Pocket ◽

Dimensional Structure ◽

Ligand Interaction ◽

Analgesic Effects ◽

Interaction Sites ◽

Potential Ligand

The landmark paper discussed in this chapter describes the crystal structure of the μ‎-opioid receptor (also known as MOP-1). Opioids are some of the oldest known drugs and have been used for over 4,000 years; however, in addition to having beneficial analgesic effects, they are associated with a myriad of side effects that can minimize their use. Although the gene sequences of the opioid receptors were determined in the 1990s it has taken much longer to translate this into visualizing their three-dimensional structure. The μ‎-opioid receptor consists of seven transmembrane α‎-helices that are connected by three extracellular loops and three intracellular loops, with a wide open binding pocket which offers many potential ligand interaction sites, and evidence of dimerization. Understanding the crystal structure of the μ‎-opioid receptor in much more detail aids explanation of the molecular determinants of ligand recognition and selectivity and will be of use in designing novel opioids with improved efficacy and fewer side effects.

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The crystal structure of Escherichia coli spermidine synthase SpeE reveals a unique substrate-binding pocket

Journal of Structural Biology ◽

10.1016/j.jsb.2009.12.024 ◽

2010 ◽

Vol 169 (3) ◽

pp. 277-285 ◽

Cited By ~ 12

Author(s):

Xingding Zhou ◽

Teck Khiang Chua ◽

Karolina L. Tkaczuk ◽

Janusz M. Bujnicki ◽

J. Sivaraman

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Substrate Binding ◽

Binding Pocket ◽

Spermidine Synthase ◽

Substrate Binding Pocket

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Crystal Structure of the Carbon-Phosphorus Lyase Complex from Escherichia coli

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314089396 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1060-C1060

Author(s):

Paulina Seweryn ◽

Lan Van ◽

Morten Kjeldgaard ◽

Bjarne Hove-Jensen ◽

Bjarne Jochimsen ◽

...

Keyword(s):

Crystal Structure ◽

Bond Cleavage ◽

Essential Element ◽

Degradation Pathway ◽

Enzyme Mechanism ◽

Radical Mechanism ◽

Stable Carbon ◽

Number Of Microorganisms ◽

Carbon Phosphorus Lyase ◽

Insight Into

Phosphorus is an essential element for all living cells and is usually taken up in the form of phosphate. A number of microorganisms, however, are capable of extracting phosphorous from organic phosphonate compounds, which are characterized by a stable carbon-phosphorus (C-P) bond (1). The metabolic pathway responsible for phosphonate degradation is still poorly understood, but the process is known to involve two reactions before the actual C-P bond cleavage, which has been proposed to take place via a radical mechanism. A key component in the process is C-P lyase, an enzyme encoded by phnJ within the phn operon (2). To get a better insight into the mechanism of this complex degradation pathway, we have determined the crystal structure of the core of a multi-subunit enzymatic complex including the C-P lyase component with a total molecular mass of 220 kDa (3). The structure reveals the overall architecture of the C-P lyase and has important implications for our understanding of enzyme mechanism and catalysis.

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