Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

2017 ◽  
Vol 73 (12) ◽  
pp. 970-984 ◽  
Author(s):  
Shenyuan Xu ◽  
Brian N. Long ◽  
Gabriel H. Boris ◽  
Anqi Chen ◽  
Shuisong Ni ◽  
...  
Keyword(s):  
Transition State ◽  
Binding Pocket ◽  
High Energy ◽  
Side Chain ◽  
Conformation Change ◽  
Gtp Hydrolysis ◽  
Structural Insight ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Intrinsic Gtpase Activity

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

scholarly journals Structure of a Talaromyces pinophilus GH62 arabinofuranosidase in complex with AraDNJ at 1.25 Å resolution

2018 ◽  
Vol 74 (8) ◽  
pp. 490-495 ◽  
Author(s):  
Olga V. Moroz ◽  
Lukasz F. Sobala ◽  
Elena Blagova ◽  
Travis Coyle ◽  
Wei Peng ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulosic Biomass ◽  
Structural Basis ◽  
Side Chain ◽  
Polymeric Substrates ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Positively Charged ◽  
Talaromyces Pinophilus ◽  
Resolution Structure

The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a K d of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.

scholarly journals The X-Ray Structure of the Haloalcohol Dehalogenase HheA from Arthrobacter sp. Strain AD2: Insight into Enantioselectivity and Halide Binding in the Haloalcohol Dehalogenase Family

2006 ◽  
Vol 188 (11) ◽  
pp. 4051-4056 ◽  
Author(s):  
René M. de Jong ◽  
Kor H. Kalk ◽  
Lixia Tang ◽  
Dick B. Janssen ◽  
Bauke W. Dijkstra
Keyword(s):  
Tertiary Structure ◽  
Halogen Bond ◽  
Environmental Pollutants ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Side Chain ◽  
Structural Determinants ◽  
Substrate Binding Pocket ◽  
X Ray ◽  
Insight Into

ABSTRACT Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheAAD2 from Arthrobacter sp. strain AD2 at 2.0-Å resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 shows that HheAAD2 has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheAAD2 can bind both enantiomers of the haloalcohol substrate 1-p-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheAAD2. Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheAAD2, in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.

scholarly journals Substituted polyfluoroaryl interactions with an arginine side chain in galectin-3 are governed by steric-, desolvation and electronic conjugation effects

2019 ◽  
Vol 17 (5) ◽  
pp. 1081-1089 ◽  
Author(s):  
Rohit Kumar ◽  
Kristoffer Peterson ◽  
Majda Misini Ignjatović ◽  
Hakon Leffler ◽  
Ulf Ryde ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Affinity ◽  
Binding Sites ◽  
Binding Pocket ◽  
Side Chain ◽  
Complex Interplay ◽  
Protein Binding Sites ◽  
Arginine Side Chain ◽  
Galectin 3 ◽  
Insight Into

Analysis of a ligand induced-aglycone-binding pocket in galectin-3 provides detailed insight into interactions of fluorinated phenyl moieties with arginine-containing protein binding sites and the complex interplay of different energetic components in defining the binding affinity.

Why do the Togni reagent and some of its derivatives exist in the high-energy hypervalent iodine form? New insight into the origins of their kinetic stability

2017 ◽  
Vol 19 (48) ◽  
pp. 32179-32183 ◽  
Author(s):  
Shungo Koichi ◽  
Benjamin Leuthold ◽  
Hans P. Lüthi
Keyword(s):  
Transition State ◽  
Thermodynamic Stability ◽  
High Energy ◽  
Kinetic Stability ◽  
Hypervalent Iodine ◽  
Insight Into

A scheme for the prediction of the kinetic and thermodynamic stability of Togni-type reagents is presented. The scheme is evaluated based on computations of the isomerization and transition state energies of an array of more than 600 compounds.

scholarly journals Structures of human mitofusin 1 provide insight into mitochondrial tethering

2016 ◽  
Vol 215 (5) ◽  
pp. 621-629 ◽  
Author(s):  
Yuanbo Qi ◽  
Liming Yan ◽  
Caiting Yu ◽  
Xiangyang Guo ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Distal Part ◽  
Binding Pocket ◽  
Magnesium Ion ◽  
Gtpase Activity ◽  
Mitochondrial Membranes ◽  
Gtpase Domain ◽  
Gtp Hydrolysis ◽  
Mitofusin 1 ◽  
Insight Into

Mitochondria undergo fusion and fission. The merging of outer mitochondrial membranes requires mitofusin (MFN), a dynamin-like GTPase. How exactly MFN mediates membrane fusion is poorly understood. Here, we determined crystal structures of a minimal GTPase domain (MGD) of human MFN1, including the predicted GTPase and the distal part of the C-terminal tail (CT). The structures revealed that a helix bundle (HB) formed by three helices extending from the GTPase and one extending from the CT closely attaches to the GTPase domain, resembling the configuration of bacterial dynamin-like protein. We show that the nucleotide-binding pocket is shallow and narrow, rendering weak hydrolysis and less dependence on magnesium ion, and that association of HB affects GTPase activity. MFN1 forms a dimer when GTP or GDP/BeF3−, but not GDP or other analogs, is added. In addition, clustering of vesicles containing membrane-anchored MGD requires continuous GTP hydrolysis. These results suggest that MFN tethers apposing membranes, likely through nucleotide-dependent dimerization.

scholarly journals Structural Insights into Inhibition of the Acinetobacter-Derived Cephalosporinase ADC-7 by Ceftazidime and Its Boronic Acid Transition State Analog

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Brandy N. Curtis ◽  
Kali A. Smolen ◽  
Sara J. Barlow ◽  
Emilia Caselli ◽  
Fabio Prati ◽  
...  
Keyword(s):  
Transition State ◽  
Boronic Acid ◽  
Carbonyl Oxygen ◽  
Hydroxyl Group ◽  
Side Chain ◽  
Enzyme Complex ◽  
Content Type ◽  
Transition State Analog ◽  
Hydrolysis Of ◽  
Structural Insights

ABSTRACT Extended-spectrum class C β-lactamases have evolved to rapidly inactivate expanded-spectrum cephalosporins, a class of antibiotics designed to be resistant to hydrolysis by β-lactamase enzymes. To better understand the mechanism by which Acinetobacter-derived cephalosporinase-7 (ADC-7), a chromosomal AmpC enzyme, hydrolyzes these molecules, we determined the X-ray crystal structure of ADC-7 in an acyl-enzyme complex with the cephalosporin ceftazidime (2.40 Å) as well as in complex with a boronic acid transition state analog inhibitor that contains the R1 side chain of ceftazidime (1.67 Å). In the acyl-enzyme complex, the carbonyl oxygen is situated in the oxyanion hole where it makes key stabilizing interactions with the main chain nitrogens of Ser64 and Ser315. The boronic acid O1 hydroxyl group is similarly positioned in this area. Conserved residues Gln120 and Asn152 form hydrogen bonds with the amide group of the R1 side chain in both complexes. These complexes represent two steps in the hydrolysis of expanded-spectrum cephalosporins by ADC-7 and offer insight into the inhibition of ADC-7 by ceftazidime through displacement of the deacylating water molecule as well as blocking its trajectory to the acyl carbonyl carbon. In addition, the transition state analog inhibitor, LP06, was shown to bind with high affinity to ADC-7 (Ki, 50 nM) and was able to restore ceftazidime susceptibility, offering the potential for optimization efforts of this type of inhibitor.

scholarly journals Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

2020 ◽  
Author(s):  
Laine Lysyk ◽  
Raelynn Brassard ◽  
Elena Arutyunova ◽  
Verena Siebert ◽  
Zhenze Jiang ◽  
...  
Keyword(s):  
Catalytic Properties ◽  
Side Chain ◽  
Turnover Rates ◽  
Rhomboid Protease ◽  
Mitochondrial Homeostasis ◽  
Substrate Preferences ◽  
Rhomboid Proteases ◽  
Hydrolysis Of ◽  
Insight Into

AbstractThe rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min−1. PGAM5 is cleaved with the highest efficiency compared to PINK1 and Smac. In proteoliposomes, a truncated β-cleavage form of PARL is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac. Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences.

scholarly journals The family 42 carbohydrate-binding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

2006 ◽  
Vol 399 (3) ◽  
pp. 503-511 ◽  
Author(s):  
Akimasa Miyanaga ◽  
Takuya Koseki ◽  
Yozo Miwa ◽  
Yuichiro Mese ◽  
Sachiko Nakamura ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Side Chain ◽  
Titration Calorimetry ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Assays ◽  
The Family ◽  
Affinity Gel Electrophoresis ◽  
Hydrolysis Of

α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.

scholarly journals Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Taylor B Updegrove ◽  
Jailynn Harke ◽  
Vivek Anantharaman ◽  
Jin Yang ◽  
Nikhil Gopalan ◽  
...  
Keyword(s):  
Active Site ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Gtpase Activity ◽  
Biological Functions ◽  
Gtp Hydrolysis ◽  
Nucleotide Base ◽  
Nucleoside Triphosphates ◽  
Nucleotide Specificity ◽  
Hydrolysis Of

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.

Modeling the Effects of O-sulfonation on the CID of Serine

2020 ◽  
Author(s):  
Kenneth Lucas ◽  
George Barnes
Keyword(s):  
Sulfo Group ◽  
High Energy ◽  
Empirical Method ◽  
Side Chain ◽  
Energy Structure ◽  
Direct Dynamics ◽  
Theoretical Mass ◽  
Intermolecular Proton Transfer ◽  
Dynamics Simulations ◽  
Semi Empirical

We present the results of direct dynamics simulations and DFT calculations aimed at elucidating the effect of \textit{O}-sulfonation on the collision induced dissociation for serine. Towards this end, direct dynamics simulations of both serine and sulfoserine were performed at multiple collision energies and theoretical mass spectra obtained. Comparisons to experimental results are favorable for both systems. Peaks related to the sulfo group are identified and the reaction dynamics explored. In particular, three significant peaks (m\z 106, 88, and 81) seen in the theoretical mass spectrum directly related to the sulfo group are analyzed as well as major peaks shared by both systems. Our analysis shows that the m\z 106 peaks result from intramolecular rearrangements, intermolecular proton transfer among complexes composed of initial fragmentation products, and at high energy side-chain fragmentation. The \mz 88 peak was found to contain multiple constitutional isomers, including a previously unconsidered, low energy structure. It was also seen that the RM1 semi empirical method was not able to obtain all of the major peaks seen in experiment for sulfoserine. In contrast, PM6 did obtain all major experimental peaks.

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