scholarly journals Analysis of the crystal structure of an active MCM hexamer

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Justin M Miller ◽  
Buenafe T Arachea ◽  
Leslie B Epling ◽  
Eric J Enemark
Keyword(s):  
Crystal Structure ◽  
Atp Hydrolysis ◽  
Pyrococcus Furiosus ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Binding Motif ◽  
Atpase Domain ◽  
Ssdna Binding ◽  
Allosteric Communication ◽  
Mcm Helicase

In a previous Research article (<xref ref-type="bibr" rid="bib25">Froelich et al., 2014</xref>), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

scholarly journals A conserved MCM single-stranded DNA binding element is essential for replication initiation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Clifford A Froelich ◽  
Sukhyun Kang ◽  
Leslie B Epling ◽  
Stephen P Bell ◽  
Eric J Enemark
Keyword(s):  
Pyrococcus Furiosus ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Binding Motif ◽  
Replication Origins ◽  
Dna Interactions ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Helicase Loading ◽  
Mcm Helicase

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation.

scholarly journals Attempts to develop an enzyme converting DHIV to KIV

2019 ◽  
Vol 32 (6) ◽  
pp. 261-270
Author(s):  
Kenji Oki ◽  
Frederick S Lee ◽  
Stephen L Mayo
Keyword(s):  
Protein Design ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Mandelate Racemase ◽  
Acid Dehydratase ◽  
Enolase Superfamily ◽  
Beta Elimination ◽  
Cost Efficient ◽  
Hydrolysis Activity

Abstract Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.

scholarly journals The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

2011 ◽  
Vol 436 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Li Phing Liew ◽  
Stephen D. Bell
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Sulfolobus Solfataricus ◽  
Active Form ◽  
Dna Helicase ◽  
Atpase Domain ◽  
Minichromosome Maintenance ◽  
Conserved Residues ◽  
Mcm Helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.

scholarly journals 1.55 Å resolution X-ray crystal structure of Rv3902c fromMycobacterium tuberculosis

2014 ◽  
Vol 70 (4) ◽  
pp. 414-417 ◽  
Author(s):  
Bharat G. Reddy ◽  
Derek B. Moates ◽  
Heung-Bok Kim ◽  
Todd J. Green ◽  
Chang-Yub Kim ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Main Chain ◽  
Binding Pocket ◽  
Bacterial Pathogenesis ◽  
Crystallographic Structure ◽  
Binding Motif ◽  
Structural Homology ◽  
X Ray ◽  
Β Sheets

The crystallographic structure of theMycobacterium tuberculosis(TB) protein Rv3902c (176 residues; molecular mass of 19.8 kDa) was determined at 1.55 Å resolution. The function of Rv3902c is unknown, although several TB genes involved in bacterial pathogenesis are expressed from the operon containing the Rv3902c gene. The unique structural fold of Rv3902c contains two domains, each consisting of antiparallel β-sheets and α-helices, creating a hand-like binding motif with a small binding pocket in the palm. Structural homology searches reveal that Rv3902c has an overall structure similar to that of theSalmonellavirulence-factor chaperone InvB, with an r.m.s.d. for main-chain atoms of 2.3 Å along an aligned domain.

scholarly journals A novel N-terminal extension in mitochondrial TRAP1 serves as a thermal regulator of chaperone activity

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
James R Partridge ◽  
Laura A Lavery ◽  
Daniel Elnatan ◽  
Nariman Naber ◽  
Roger Cooke ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Temperature Dependence ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Chaperone Activity ◽  
Regulatory Function ◽  
Protein Homeostasis ◽  
Closed State ◽  
Higher Eukaryotes ◽  
Rate Limiting

Hsp90 is a conserved chaperone that facilitates protein homeostasis. Our crystal structure of the mitochondrial Hsp90, TRAP1, revealed an extension of the N-terminal β-strand previously shown to cross between protomers in the closed state. In this study, we address the regulatory function of this extension or ‘strap’ and demonstrate its responsibility for an unusual temperature dependence in ATPase rates. This dependence is a consequence of a thermally sensitive kinetic barrier between the apo ‘open’ and ATP-bound ‘closed’ conformations. The strap stabilizes the closed state through trans-protomer interactions. Displacement of cis-protomer contacts from the apo state is rate-limiting for closure and ATP hydrolysis. Strap release is coupled to rotation of the N-terminal domain and dynamics of the nucleotide binding pocket lid. The strap is conserved in higher eukaryotes but absent from yeast and prokaryotes suggesting its role as a thermal and kinetic regulator, adapting Hsp90s to the demands of unique cellular and organismal environments.

scholarly journals Crystal structure of unusual RNA duplex containing strontium ion binding motif

2014 ◽  
Vol 70 (a1) ◽  
pp. C1378-C1378
Author(s):  
Hiroki Kanazawa ◽  
Jiro Kondo
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Base Pair ◽  
Binding Pocket ◽  
Minor Groove ◽  
Diffusion Method ◽  
Ion Binding ◽  
Binding Motif ◽  
Base Pairs ◽  
Strontium Ion

Crystal structures of several functional non-coding RNAs, such as ribozymes, aptamers, ribosomes and tRNAs, have been reported so far. Unusual structural motifs and non-complementary base pairs are important for their functions. In the present study, we have determined a crystal structure of an unusual RNA duplex containing a strontium ion binding motif. A 19 mer RNA (5'-UUGUCGCUU[Br]CGAAAAAGUC-3') was chemical synthesized and purified by denaturing PAGE. Crystallizations were performed by the sitting-drop vapor diffusion method. The initial phase was solved by the SAD method. Atomic parameters were refined at a resolution of 3.0 Å. The 19 mer RNA forms an unusual antiparallel duplex. At both ends of the duplex, the Watson-Crick G=C and A-U and the Wobble GoU and AoC base pairs are formed. The Wobble C10oA14* pair is available only in acidic condition by protonation of N1 of A14* (* indicates residues of the opposite strand). Two hydrogen bonds, N1-H(A14*)...O2(C10) and N6-H(A14*)...N3(C10), are observed in the base pair. In the center of the duplex, two sheared G11oA13* and G11*oA13 base pairs are formed. The distance between two RNA chains becomes shorter by the GoA base pair and hydrogen bonds between the Watson-Crick edge of G11 and the phosphate group of A12*. Therefore, the central A12 residue cannot make a base pair, but it makes a stacking interaction with A12*. The A12 residue stacks also with A13 of the sheared GoA base pair. As a result, an A13-A12-A12*-A13* stacked column is formed at the minor groove of the duplex, and the G11 base of the sheared GoA base pair is inclined toward the minor groove. By taking such a unique structure, the RNA duplex has a Sr2+ ion binding pocket in the center. A hydrated Sr2+ ion binds to O6 and N7 of G11 and G11*. The Sr2+ ion is surrounded by four phosphate groups of two RNA chains. The Sr2+ ion is tightly captured by eight hydrogen bonds in total.

scholarly journals ATP hydrolysis by the SNF2 domain of the ultraspecific maintenance methylase Dnmt5 drives recognition and modification of hemimethylated DNA

2020 ◽  
Author(s):  
Phillip A. Dumesic ◽  
Caitlin I. Stoddard ◽  
Sandra Catania ◽  
Geeta J. Narlikar ◽  
Hiten D. Madhani
Keyword(s):  
Dna Methylation ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Substrate Recognition ◽  
Binding Pocket ◽  
Atpase Domain ◽  
Base Flipping ◽  
Kinetic Proofreading ◽  
Exquisite Specificity

SUMMARYC. neoformans Dnmt5 is an ultraspecific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step that is essential for DNA methylation. Such coupling occurs independently of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating the Dnmt5 ATP binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a ‘flipped-out’ conformation of the target cytosine bypass the SNF2 domain’s requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to base-flipping by the DNMT domain. These findings establish a new role for a SNF2 ATPase domain: controlling substrate recognition and catalysis by an adjoined enzymatic domain. This coupling may contribute to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

scholarly journals Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft

2019 ◽  
Vol 295 (6) ◽  
pp. 1551-1564 ◽  
Author(s):  
Kelly E. Du Pont ◽  
Russell B. Davidson ◽  
Martin McCullagh ◽  
Brian J. Geiss
Keyword(s):  
Molecular Dynamics ◽  
Structural Changes ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Binding Pocket ◽  
Energy Transduction ◽  
Helicase Domain ◽  
Genome Replication ◽  
Turnover Rates

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential “communication hub” for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

scholarly journals Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

2005 ◽  
Vol 187 (7) ◽  
pp. 2386-2394 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Andrea Gorrell ◽  
Sarah H. Lawrence ◽  
Prabha Iyer ◽  
Kerry Smith ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Binding Pocket ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Acetyl Phosphate ◽  
Hydrophobic Pocket ◽  
Methanosarcina Thermophila ◽  
Methyl Group ◽  
Substrate Binding Sites

ABSTRACT Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val93, Leu122, Phe179, and Pro232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with Km values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

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