Crystal Structure of Inhibitor-Bound P450BM-3 Reveals Open Conformation of Substrate Access Channel†,‡

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb–LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

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Crystal structure of enolase fromDrosophila melanogaster

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17004022 ◽

2017 ◽

Vol 73 (4) ◽

pp. 228-234 ◽

Cited By ~ 2

Author(s):

Congcong Sun ◽

Baokui Xu ◽

Xueyan Liu ◽

Zhen Zhang ◽

Zhongliang Su

Keyword(s):

Crystal Structure ◽

Catalytic Activity ◽

Structural Basis ◽

Molecular Replacement ◽

Biological Processes ◽

Conserved Residues ◽

Dimer Interface ◽

Open Conformation ◽

Evolutionary Studies ◽

Important Enzyme

Enolase is an important enzyme in glycolysis and various biological processes. Its dysfunction is closely associated with diseases. Here, the enolase fromDrosophila melanogaster(DmENO) was purified and crystallized. A crystal of DmENO diffracted to 2.0 Å resolution and belonged to space groupR32. The structure was solved by molecular replacement. Like most enolases, DmENO forms a homodimer with conserved residues in the dimer interface. DmENO possesses an open conformation in this structure and contains conserved elements for catalytic activity. This work provides a structural basis for further functional and evolutionary studies of enolase.

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Crystal Structure ofPseudomonas aeruginosaLipase in the Open Conformation

Journal of Biological Chemistry ◽

10.1074/jbc.m003903200 ◽

2000 ◽

Vol 275 (40) ◽

pp. 31219-31225 ◽

Cited By ~ 185

Author(s):

Marco Nardini ◽

Dietmar A. Lang ◽

Klaus Liebeton ◽

Karl-Erich Jaeger ◽

Bauke W. Dijkstra

Keyword(s):

Crystal Structure ◽

Open Conformation

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An Open Conformation of Switch I Revealed by the Crystal Structure of a Mg2+-free Form of RHOA Complexed with GDP

Journal of Biological Chemistry ◽

10.1074/jbc.m910274199 ◽

2000 ◽

Vol 275 (24) ◽

pp. 18311-18317 ◽

Cited By ~ 40

Author(s):

Toshiyuki Shimizu ◽

Kentaro Ihara ◽

Ryoko Maesaki ◽

Shinya Kuroda ◽

Kozo Kaibuchi ◽

...

Keyword(s):

Crystal Structure ◽

Free Form ◽

Open Conformation

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Structure of the Ca2+-saturated C-terminal domain of scallop troponin C in complex with a troponin I fragment

Biological Chemistry ◽

10.1515/hsz-2012-0152 ◽

2013 ◽

Vol 394 (1) ◽

pp. 55-68 ◽

Cited By ~ 5

Author(s):

Yusuke S. Kato ◽

Fumiaki Yumoto ◽

Hiroyuki Tanaka ◽

Takuya Miyakawa ◽

Yumiko Miyauchi ◽

...

Keyword(s):

Crystal Structure ◽

Structural Change ◽

Troponin I ◽

Adductor Muscle ◽

Troponin C ◽

Striated Muscles ◽

Terminal Domain ◽

Open Conformation ◽

Ef Hands ◽

Contractile Forces

Abstract Troponin C (TnC) is the Ca2+-sensing subunit of troponin that triggers the contraction of striated muscles. In scallops, the striated muscles consume little ATP energy in sustaining strong contractile forces. The N-terminal domain of TnC works as the Ca2+ sensor in vertebrates, whereas scallop TnC uses the C-terminal domain as the Ca2+ sensor, suggesting that there are differences in the mechanism of the Ca2+-dependent regulation of muscles between invertebrates and vertebrates. Here, we report the crystal structure of Akazara scallop (Chlamys nipponensis akazara) adductor muscle TnC C-terminal domain (asTnCC) complexed with a short troponin I fragment (asTnIS) and Ca2+. The electron density of a Ca2+ ion is observed in only one of the two EF-hands. The EF-hands of asTnCC can only be in the fully open conformation with the assistance of asTnIS. The number of hydrogen bonds between asTnCC and asTnIS is markedly lower than the number in the vertebrate counterparts. The Ca2+ modulation on the binding between asTnCC and asTnIS is weaker, but structural change of the complex depending on Ca2+ concentration was observed. Together, these findings provide a detailed description of the distinct molecular mechanism of contractile regulation in the scallop adductor muscle from that of vertebrates.

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Crystal structure of the RNA 2′,3′-cyclic phosphodiesterase fromDeinococcus radiodurans

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17004964 ◽

2017 ◽

Vol 73 (5) ◽

pp. 276-280 ◽

Cited By ~ 1

Author(s):

Wanchun Han ◽

Jiahui Cheng ◽

Congli Zhou ◽

Yuejin Hua ◽

Ye Zhao

Keyword(s):

Crystal Structure ◽

Active Site ◽

Catalytic Mechanism ◽

Deinococcus Radiodurans ◽

Sulfate Ion ◽

Open Conformation ◽

Domains Of Life

2′,3′-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase fromDeinococcus radioduranswas crystallized and the crystals diffracted to 1.6 Å resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.

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Crystal Structures of [Fe]-Hydrogenase from Methanolacinia paynteri Suggest a Path of the FeGP-Cofactor Incorporation Process

Inorganics ◽

10.3390/inorganics8090050 ◽

2020 ◽

Vol 8 (9) ◽

pp. 50

Author(s):

Gangfeng Huang ◽

Francisco Javier Arriaza-Gallardo ◽

Tristan Wagner ◽

Seigo Shima

Keyword(s):

Crystal Structure ◽

Protein Structure ◽

Guanosine Monophosphate ◽

Amino Group ◽

Prosthetic Group ◽

Closed Conformation ◽

Open Conformation ◽

Local Protein Structure ◽

Local Protein

[Fe]-hydrogenase (Hmd) catalyzes the reversible heterolytic cleavage of H2, and hydride transfer to methenyl-tetrahydromethanopterin (methenyl-H4MPT+). The iron-guanylylpyridinol (FeGP) cofactor, the prosthetic group of Hmd, can be extracted from the holoenzyme and inserted back into the protein. Here, we report the crystal structure of an asymmetric homodimer of Hmd from Methanolacinia paynteri (pHmd), which was composed of one monomer in the open conformation with the FeGP cofactor (holo-form) and a second monomer in the closed conformation without the cofactor (apo-form). In addition, we report the symmetric pHmd-homodimer structure in complex with guanosine monophosphate (GMP) or guanylylpyridinol (GP), in which each ligand was bound to the protein, where the GMP moiety of the FeGP-cofactor is bound in the holo-form. Binding of GMP and GP modified the local protein structure but did not induce the open conformation. The amino-group of the Lys150 appears to interact with the 2-hydroxy group of pyridinol ring in the pHmd–GP complex, which is not the case in the structure of the pHmd–FeGP complex. Lys150Ala mutation decreased the reconstitution rate of the active enzyme with the FeGP cofactor at the physiological pH. These results suggest that Lys150 might be involved in the FeGP-cofactor incorporation into the Hmd protein in vivo.

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Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714022755 ◽

2014 ◽

Vol 70 (12) ◽

pp. 3253-3265 ◽

Cited By ~ 21

Author(s):

Elena Fernández-Fueyo ◽

Sandra Acebes ◽

Francisco J. Ruiz-Dueñas ◽

María Jesús Martínez ◽

Antonio Romero ◽

...

Keyword(s):

Crystal Structure ◽

Electron Transfer ◽

Manganese Peroxidase ◽

Catalytic Properties ◽

Substrate Oxidation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Ceriporiopsis Subvermispora ◽

Ligninolytic Fungi ◽

Access Channel

The genome ofCeriporiopsis subvermisporaincludes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+oxidation by the internal propionate, but prevents the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of anin silicoshortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

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