Crystal structures and catalytic mechanism of theC-methyltransferase Coq5 provide insights into a key step of the yeast coenzyme Q synthesis pathway

Saccharomyces cerevisiaeCoq5 is anS-adenosyl methionine (SAM)-dependent methyltransferase (SAM-MTase) that catalyzes the onlyC-methylation step in the coenzyme Q (CoQ) biosynthesis pathway, in which 2-methoxy-6-polyprenyl-1,4-benzoquinone (DDMQH2) is converted to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone (DMQH2). Crystal structures of Coq5 were determined in the apo form (Coq5-apo) at 2.2 Å resolution and in the SAM-bound form (Coq5-SAM) at 2.4 Å resolution, representing the first pair of structures for the yeast CoQ biosynthetic enzymes. Coq5 displays a typical class I SAM-MTase structure with two minor variations beyond the core domain, both of which are considered to participate in dimerization and/or substrate recognition. Slight conformational changes at the active-site pocket were observed upon binding of SAM. Structure-based computational simulation using an analogue of DDMQH2enabled us to identify the binding pocket and entrance tunnel of the substrate. Multiple-sequence alignment showed that the residues contributing to the dimeric interface and the SAM- and DDMQH2-binding sites are highly conserved in Coq5 and homologues from diverse species. A putative catalytic mechanism of Coq5 was proposed in which Arg201 acts as a general base to initiate catalysis with the help of a water molecule.

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Pyrethroid Carboxylesterase PytH from Sphingobium faniae JZ-2: Structure and Catalytic Mechanism

Applied and Environmental Microbiology ◽

10.1128/aem.02971-19 ◽

2020 ◽

Vol 86 (12) ◽

Cited By ~ 2

Author(s):

Dongqing Xu ◽

Yanyan Gao ◽

Bo Sun ◽

Tingting Ran ◽

Liangping Zeng ◽

...

Keyword(s):

Crystal Structures ◽

Catalytic Mechanism ◽

Hydrophobic Pocket ◽

Core Domain ◽

Content Type ◽

Sequence Identity ◽

Hydrolase Fold ◽

Wide Range ◽

Pyrethroid Pesticides ◽

Lid Domain

ABSTRACT Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/β-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates. IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/β-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/β-hydrolase members.

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Crystal structures of Bax and Bak Reveal Molecular Events Initiating Apoptosis

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331408509x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1490-C1490

Author(s):

Peter Czabotar ◽

Jason Brouwer ◽

Dana Westphal ◽

Geoff Thompson ◽

Peter Colman

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Mitochondrial Membrane ◽

Molecular Mechanisms ◽

Structural Studies ◽

Core Domain ◽

Hydrophobic Groove ◽

Outer Leaflet ◽

Molecular Events ◽

Surface Crystal

A key event in apoptosis is the conversion of Bax or Bak from inert monomers into cytotoxic mitochondrial membrane perforating oligomers. Certain BH3-only relatives can initiate this step through direct interactions, yet the means by which conformational changes are invoked, the nature of the conformational changes themselves, the mechanism by which they insert into membranes and the process by which they perforate these barriers has largely remained a mystery. Our recent structural studies provided the first insights into this process for Bax [1]. We found that BH3 domains activate Bax by binding to a hydrophobic groove on its surface. Crystal structures of these complexes revealed an unexpected conformational change involving dissociation of a previously unrecognized "core" domain from a "latch' domain. A further structure of the freed Bax "core" domains revealed that these form dimers that possess a surface of aromatic residues which we hypothesis engage the outer leaflet of the mitochondrial membrane and induce curvature. We have now extended our studies to include structures of Bax bound to alternative BH3-only proteins providing new insights into key interactions occurring at this interface. Additionally, we have solved structures of activated Bak and of the freed Bak "core" domain dimers. These results further our understanding of the molecular mechanisms by which these highly dynamic proteins engage the mitochondrial membrane and thus control the life/death switch in cells.

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Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

10.26434/chemrxiv.14403929.v1 ◽

2021 ◽

Author(s):

Lee M Stunkard ◽

Aaron B Benjamin ◽

James Bower ◽

Tyler Huth ◽

Jeremy Lohman

Keyword(s):

Transition State ◽

Crystal Structures ◽

Conformational Changes ◽

Catalytic Mechanism ◽

Streptomyces Coelicolor ◽

Base Catalysis ◽

Acid Base ◽

Transition State Analog ◽

Catalytic Residues ◽

General Acid

Crystal structures of Streptomyces coelicolor methylmalonyl-CoA epimerase in the holo-form, with substrate or the putative transition state analog, 2-nitroproionyl-CoA. The proposed catalytic mechanism is general acid-base catalysis. The proposed catalytic residues are too far from the substrate or analog, unless conformational changes take place or some other mechanism is used. <br>

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Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein

10.1101/2021.04.02.438172 ◽

2021 ◽

Author(s):

Swagatha Ghosh ◽

Sayan Mondal ◽

Keerti Yadav ◽

Shantanu Aggarwal ◽

Wayne F. Schaefer ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Spectral Properties ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Binding Pocket ◽

Structural Heterogeneity ◽

Wild Type ◽

Absorbing States

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates. Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm. Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin (~18kDa) binds BLA with similar affinities and show a broad red- shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA- bound lipocalins, known only in phytochromes till date.

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The HIV-1 Env gp120 Inner Domain Shapes the Phe43 Cavity and the CD4 Binding Site

mBio ◽

10.1128/mbio.00280-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Jérémie Prévost ◽

William D. Tolbert ◽

Halima Medjahed ◽

Rebekah T. Sherburn ◽

Navid Madani ◽

...

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Conformational Changes ◽

Binding Pocket ◽

Envelope Glycoproteins ◽

Complex Interplay ◽

Structural Determinants ◽

Gp120 Core ◽

Cd4 Binding Site ◽

Hiv 1

ABSTRACT The HIV-1 envelope glycoproteins (Env) undergo conformational changes upon interaction of the gp120 exterior glycoprotein with the CD4 receptor. The gp120 inner domain topological layers facilitate the transition of Env to the CD4-bound conformation. CD4 engages gp120 by introducing its phenylalanine 43 (Phe43) in a cavity (“the Phe43 cavity”) located at the interface between the inner and outer gp120 domains. Small CD4-mimetic compounds (CD4mc) can bind within the Phe43 cavity and trigger conformational changes similar to those induced by CD4. Crystal structures of CD4mc in complex with a modified CRF01_AE gp120 core revealed the importance of these gp120 inner domain layers in stabilizing the Phe43 cavity and shaping the CD4 binding site. Our studies reveal a complex interplay between the gp120 inner domain and the Phe43 cavity and generate useful information for the development of more-potent CD4mc. IMPORTANCE The Phe43 cavity of HIV-1 envelope glycoproteins (Env) is an attractive druggable target. New promising compounds, including small CD4 mimetics (CD4mc), were shown to insert deeply into this cavity. Here, we identify a new network of residues that helps to shape this highly conserved CD4 binding pocket and characterize the structural determinants responsible for Env sensitivity to small CD4 mimetics.

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Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

10.26434/chemrxiv.14403929 ◽

2021 ◽

Author(s):

Lee M Stunkard ◽

Aaron B Benjamin ◽

James Bower ◽

Tyler Huth ◽

Jeremy Lohman

Keyword(s):

Transition State ◽

Crystal Structures ◽

Conformational Changes ◽

Catalytic Mechanism ◽

Streptomyces Coelicolor ◽

Base Catalysis ◽

Acid Base ◽

Transition State Analog ◽

Catalytic Residues ◽

General Acid

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2

Nature Communications ◽

10.1038/s41467-021-22731-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qing-Tao He ◽

Peng Xiao ◽

Shen-Ming Huang ◽

Ying-Li Jia ◽

Zhong-Liang Zhu ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Nmr Spectrum ◽

H Nmr ◽

Interaction Sites ◽

Receptor Interactions ◽

Remote Sites ◽

Genetic Code Expansion ◽

G Protein Coupled ◽

Interaction Change

AbstractArrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.

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Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

Biochemical Society Transactions ◽

10.1042/bst20150138 ◽

2015 ◽

Vol 43 (5) ◽

pp. 1023-1032 ◽

Cited By ~ 10

Author(s):

Thomas Stockner ◽

Anna Mullen ◽

Fraser MacMillan

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Abc Transporters ◽

Epr Spectroscopy ◽

Structural Information ◽

Dynamic Properties ◽

Molecular System ◽

Synergistic Effects ◽

Md Simulations ◽

Substrate Spectrum

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented.

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