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1.
IUBMB Life ; 72(6): 1233-1242, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32271995

RESUMO

C─H⋯O hydrogen bonds constitute a unique class of cohesive interactions. Their properties are similar to those of canonical H-bonds, although their energy is significantly lower, typically in the 0.5-2.5 kcal/mol range. Polarised C─H groups, such as those adjacent to electronegative groups, or within aromatic moieties, are particularly strong donors. C─H⋯O bonds are ubiquitous in nucleic acids and in proteins, notably stabilizing the ß-sheet secondary structure. They have also been observed in numerous protein-ligand interactions. Here, we analysed crystal structures, deposited in the Protein Data Bank, of complexes of FDA-approved protein kinase inhibitors with cognate kinases, to assess the possible role of C─Hinhibitor ⋯Oprotein hydrogen bonds. The conserved hinge motif of protein kinases with two solvent-exposed carbonyl groups and one exposed backbone amide, is well known to be involved in canonical H-bonding with inhibitors. We now find that in virtually all complexes where the inhibitor interacts with the hinge backbone, at least one of the hinge carbonyl groups accepts an H-bond from a C─H inhibitor group, which is either aromatic or adjacent to an electronegative group. These observations are important for design of hinge-binding scaffolds of novel kinase inhibitors for therapeutic use.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Estereoisomerismo
2.
EMBO J ; 32(7): 1023-35, 2013 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-23455152

RESUMO

Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)-based motor. Dynactin binds both dynein and MTs via its p150(Glued) subunit, but little is known about the 'pointed-end complex' that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin-dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo-like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore-MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left-handed ß-helix domain, with the phosphorylation site located within a disordered, C-terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Bovinos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Embrião de Galinha , Complexo Dinactina , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Quinase 1 Polo-Like
3.
Postepy Biochem ; 62(3): 286-297, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28132483

RESUMO

Macromolecular X-ray crystallography has undergone a dramatic and astonishing transformation since its inception in mid 1950s, almost exclusively owing to the developments in three other fields: computer science; synchrotron radiation; and molecular biology. The process of structure solution from a single crystal, provided the quality of diffraction data is adequate, has been shortened from many years to hours, if not minutes. Yet, in spite of the exponential increase in the available structural information (~120, 000 structures in the Protein Data Bank today), many fundamental problems continue to be the subject of scientific controversy. This article contains personal recollections of the author, pertaining to two research projects - conducted nearly four decades apart - both of which touch upon such long standing discussion of the Monod-Wyman-Changeux theory of cooperativity (or 'conformational selection') vs the Koshland-Nemethy-Filmer theory of 'induced fit'. It is dedicated to Dr. Alexander Wlodawer on his 70th birthday, with best wishes of continuing success.


Assuntos
Cristalografia por Raios X/métodos , Cristalografia por Raios X/história , História do Século XX , História do Século XXI , Conformação Proteica , Proteínas/química , Proteínas/metabolismo
4.
J Biol Chem ; 288(47): 34030-34040, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24106280

RESUMO

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood. Using permeabilized blood vessels from PRG(-/-) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca(2+)-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5'-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca(2+) transient and phasic force components and the onset of Ca(2+)-sensitized force.


Assuntos
Cálcio/metabolismo , Fatores de Troca do Nucleotídeo Guanina/agonistas , Guanosina 5'-O-(3-Tiotrifosfato)/análogos & derivados , Fenilefrina/análogos & derivados , Fatores de Troca de Nucleotídeo Guanina Rho/agonistas , Animais , Linhagem Celular , Inativação Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Fenilefrina/farmacologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Coelhos , Ratos , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
5.
Biochim Biophys Acta ; 1834(7): 1285-91, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23541530

RESUMO

All known protein kinases share a bilobal kinase domain with well conserved structural elements. Because of significant structural similarities of nucleotide binding pocket, the development of highly selective kinase inhibitors is a very challenging task. Flavonols, naturally occurring plant metabolites, have long been known to inhibit kinases by mimicking the adenine moiety. Interestingly, recent data show that some flavonol glycosides are more selective, although underlying mechanisms were unknown. Crystallographic data from our laboratory revealed that the N-terminal kinase domain of p90 ribosomal S6 kinase, isoform 2, binds three different flavonol rhamnosides in a highly unusual manner, distinct from other kinase inhibitor interactions. The kinase domain undergoes a reorganization of several structural elements in response to the binding of the inhibitors. Specifically, the main ß-sheet of the N-lobe undergoes a twisting rotation by ~56° around an axis passing through the N- and C-lobes, leading to the restructuring of the canonical ATP-binding pocket into pockets sterically adapted to the inhibitor shape. The flavonol rhamnosides appear to adopt compact, but strained conformations with the rhamnose moiety swept under the B-ring of flavonol, unlike the structure of the free counterparts in solution. These data suggest that the flavonol glycoside scaffold could be used as a template for new inhibitors selective for the RSK family. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).


Assuntos
Flavonóis/farmacologia , Glicosídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Cristalografia por Raios X , Flavonóis/química , Flavonóis/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Ramnose/química , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo
6.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 9): 2420-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25195755

RESUMO

Ebolavirus (EBOV) causes severe hemorrhagic fever with a mortality rate of up to 90%. EBOV is a member of the order Mononegavirales and, like other viruses in this taxonomic group, contains a negative-sense single-stranded (ss) RNA. The EBOV ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. Like other EBOV proteins, NP is multifunctional. It is tightly associated with the viral genome and is essential for viral transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. NP is unusual among the Mononegavirales in that it contains two distinct regions, or putative domains, the C-terminal of which shows no homology to any known proteins and is purported to be a hub for protein-protein interactions within the nucleocapsid. The atomic structure of NP remains unknown. Here, the boundaries of the N- and C-terminal domains of NP from Zaire EBOV are defined, it is shown that they can be expressed as highly stable recombinant proteins in Escherichia coli, and the atomic structure of the C-terminal domain (residues 641-739) derived from analysis of two distinct crystal forms at 1.98 and 1.75 Šresolution is described. The structure reveals a novel tertiary fold that is distantly reminiscent of the ß-grasp architecture.


Assuntos
Ebolavirus/química , Nucleoproteínas/química , Proteínas Virais/química , Sequência de Aminoácidos , Cristalografia por Raios X , Ebolavirus/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos
7.
J Phys Chem B ; 128(30): 7376-7384, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39024061

RESUMO

The effects of deviations from nonlinearity around the carbonyl proton acceptor of an amide group are assessed by DFT quantum chemical calculations for both CH··O and NH··O H-bonds. The proton donors are the imidazole functional group of His and the indole of Trp, which are paired respectively with N-methylacetamide and acetamide. The displacement of either CH or NH group toward the carbonyl O sp2 lone pairs stabilizes the system and strengthens the H-bond. But the two donor groups differ in their response to a shift out of the amide plane. While the NH··O H-bond is weakened by this displacement, a substantial strengthening is observed when the CH donor is moved out of this plane, in one direction versus the other. This pattern is explained on the basis of simple Coulombic considerations.


Assuntos
Ligação de Hidrogênio , Proteínas , Prótons , Proteínas/química , Teoria da Densidade Funcional , Acetamidas/química
8.
Acta Crystallogr D Struct Biol ; 80(Pt 7): 551-562, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38941144

RESUMO

Tryptophan is the most prominent amino acid found in proteins, with multiple functional roles. Its side chain is made up of the hydrophobic indole moiety, with two groups that act as donors in hydrogen bonds: the Nϵ-H group, which is a potent donor in canonical hydrogen bonds, and a polarized Cδ1-H group, which is capable of forming weaker, noncanonical hydrogen bonds. Due to adjacent electron-withdrawing moieties, C-H...O hydrogen bonds are ubiquitous in macromolecules, albeit contingent on the polarization of the donor C-H group. Consequently, Cα-H groups (adjacent to the carbonyl and amino groups of flanking peptide bonds), as well as the Cϵ1-H and Cδ2-H groups of histidines (adjacent to imidazole N atoms), are known to serve as donors in hydrogen bonds, for example stabilizing parallel and antiparallel ß-sheets. However, the nature and the functional role of interactions involving the Cδ1-H group of the indole ring of tryptophan are not well characterized. Here, data mining of high-resolution (r ≤ 1.5 Å) crystal structures from the Protein Data Bank was performed and ubiquitous close contacts between the Cδ1-H groups of tryptophan and a range of electronegative acceptors were identified, specifically main-chain carbonyl O atoms immediately upstream and downstream in the polypeptide chain. The stereochemical analysis shows that most of the interactions bear all of the hallmarks of proper hydrogen bonds. At the same time, their cohesive nature is confirmed by quantum-chemical calculations, which reveal interaction energies of 1.5-3.0 kcal mol-1, depending on the specific stereochemistry.


Assuntos
Ligação de Hidrogênio , Proteínas , Triptofano , Triptofano/química , Proteínas/química , Modelos Moleculares , Cristalografia por Raios X/métodos , Conformação Proteica
9.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 2): 266-75, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23385462

RESUMO

Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45-346) of mouse RSK2, or RSK2(NTKD), has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3'',4''-di-O-acetyl-α-L-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-L-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2(NTKD) with a dissociation constant (K(d)) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K(d) for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca(2+).


Assuntos
Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quercetina/análogos & derivados , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Animais , Cristalografia por Raios X , Camundongos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Quercetina/metabolismo , Quercetina/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Termodinâmica
10.
Circ Res ; 109(9): 993-1002, 2011 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-21885830

RESUMO

RATIONALE: In normal and diseased vascular smooth muscle (SM), the RhoA pathway, which is activated by multiple agonists through G protein-coupled receptors (GPCRs), plays a central role in regulating basal tone and peripheral resistance. This occurs through inhibition of myosin light chain phosphatase, leading to increased phosphorylation of the myosin regulatory light chain. Although it is thought that specific agonists and GPCRs may couple to distinct RhoA guanine nucleotide exchange factors (GEFs), thus raising the possibility of selective targeting of specific GEFs for therapeutic use, this notion is largely unexplored for SM contraction. OBJECTIVE: We examine whether p63RhoGEF, known to couple specifically to Gα(q/11) in vitro, is functional in blood vessels as a mediator of RhoA activation and if it is selectively activated by Gα(q/11) coupled agonists. METHODS AND RESULTS: We find that p63RhoGEF is present across SM tissues and demonstrate that silencing of the endogenous p63RhoGEF in mouse portal vein inhibits contractile force induced by endothelin-1 to a greater extent than the predominantly Gα(12/13)-mediated thromboxane analog U46619. This is because endothelin-1 acts on Gα(q/11) as well as Gα(12/13). Introduction of the exogenous isolated pleckstrin-homology (PH) domain of p63RhoGEF (residues 331-580) into permeabilized rabbit portal vein inhibited Ca2+ sensitized force and activation of RhoA, when phenylephrine was used as an agonist. This reinforces the results based on endothelin-1, because phenylephrine is thought to act exclusively through Gα(q/11). CONCLUSION: We demonstrate that p63RhoGEF selectively couples Gα(q/11) but not Gα(12/13), to RhoA activation in blood vessels and cultured cells and thus mediates the physiologically important Ca2+ sensitization of force induced with Gα(q/11)-coupled agonists. Our results suggest that signaling through p63RhoGEF provides a novel mechanism for selective regulation of blood pressure.


Assuntos
Cálcio/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Contração Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Endotelina-1/farmacologia , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Fenilefrina/farmacologia , Veia Porta/fisiologia , Coelhos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Vasoconstritores/farmacologia , Proteína rhoA de Ligação ao GTP/fisiologia
11.
Front Physiol ; 14: 1228488, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37781225

RESUMO

Introduction: Phosphorylation of smooth muscle (SM) myosin regulatory light chain (RLC20) is a critical switch leading to SM contraction. The canonical view held that only the short isoform of myosin light chain kinase (MLCK1) catalyzed this reaction. It is now accepted that auxiliary kinases may contribute to vascular SM tone and contractility. We have previously reported that p90 ribosomal S6 kinase (RSK2) functions as such a kinase, in parallel with MLCK1, contributing ∼25% of the maximal myogenic force in resistance arteries. Thus, RSK2 may be instrumental in the regulation of basal vascular tone and blood pressure. Here, we take advantage of a MLCK1 null mouse (mylk1 -/-) to further test our hypothesis that RSK2 can function as an MLCK, playing a significant physiological role in SM contractility. Methods: Using fetal (E14.5-18.5) SM tissues, as embryos die at birth, we investigated the necessity of MLCK for contractility and fetal development and determined the ability of RSK2 kinase to compensate for the lack of MLCK and characterized its signaling pathway in SM. Results and Discussion: Agonists induced contraction and RLC20 phosphorylation in mylk1 -/- SM was attenuated by RSK2 inhibition. The pCa-tension relationships in permeabilized strips of bladder showed no difference in Ca2+ sensitivity in WT vs mylk1 -/- muscles, although the magnitude of force responses was considerably smaller in the absence of MLCK. The magnitude of contractile responses was similar upon addition of GTPγS to activate the RhoA/ROCK pathway or calyculinA to inhibit the myosin phosphatase. The Ca2+-dependent tyrosine kinase, Pyk2, contributed to RSK2-mediated contractility and RLC20 phosphorylation. Proximity-ligation and immunoprecipitation assays demonstrated an association of RSK2, PDK1 and ERK1/2 with MLCK and actin. RSK2, PDK1, ERK1/2 and MLCK formed a signaling complex on the actin filament, positioning them for interaction with adjacent myosin heads. The Ca2+-dependent component reflected the agonist mediated increases in Ca2+, which activated the Pyk2/PDK1/RSK2 signaling cascade. The Ca2+-independent component was through activation of Erk1/2/PDK1/RSK2 leading to direct phosphorylation of RLC20, to increase contraction. Overall, RSK2 signaling constitutes a new third signaling pathway, in addition to the established Ca2+/CaM/MLCK and RhoA/ROCK pathways to regulate SM contractility.

12.
bioRxiv ; 2023 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-37292593

RESUMO

Background: Phosphorylation of smooth muscle (SM) myosin regulatory light chain (RLC 20 ) is a critical switch leading to contraction or cell migration. The canonical view held that the only kinase catalyzing this reaction is the short isoform of myosin light chain kinase (MLCK1). Auxiliary kinases may be involved and play a vital role in blood pressure homeostasis. We have previously reported that p90 ribosomal S6 kinase (RSK2) functions as such a kinase, in parallel with the classical MLCK1, contributing ∼25% of the maximal myogenic force in resistance arteries and regulating blood pressure. Here, we take advantage of a MLCK1 null mouse to further test our hypothesis that RSK2 can function as an MLCK, playing a significant physiological role in SM contractility. Methods: Fetal (E14.5-18.5) SM tissues were used as embryos die at birth. We investigated the necessity of MLCK for contractility, cell migration and fetal development and determined the ability of RSK2 kinase to compensate for the lack of MLCK and characterized it's signaling pathway in SM. Results: Agonists induced contraction and RLC 20 phosphorylation in mylk1 -/- SM, that was inhibited by RSK2 inhibitors. Embryos developed and cells migrated in the absence of MLCK. The pCa-tension relationships in WT vs mylk1 -/- muscles demonstrated a Ca 2+ -dependency due to the Ca 2+ -dependent tyrosine kinase Pyk2, known to activate PDK1 that phosphorylates and fully activates RSK2. The magnitude of contractile responses was similar upon addition of GTPγS to activate the RhoA/ROCK pathway. The Ca 2+ -independent component was through activation of Erk1/2/PDK1/RSK2 leading to direct phosphorylation of RLC 20 , to increase contraction. RSK2, PDK1, Erk1/2 and MLCK formed a signaling complex on the actin filament, optimally positioning them for interaction with adjacent myosin heads. Conclusions: RSK2 signaling constitutes a new third signaling pathway, in addition to the established Ca 2+ /CAM/MLCK and RhoA/ROCK pathways to regulate SM contractility and cell migration.

13.
Mol Biol Cell ; 34(1): br1, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36350697

RESUMO

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally absent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters in Xenopus extracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.


Assuntos
Dineínas , Fuso Acromático , Humanos , Dineínas/metabolismo , Fuso Acromático/metabolismo , Aurora Quinase A/metabolismo , Cinetocoros/metabolismo , Proteínas de Ciclo Celular/metabolismo , Polos do Fuso/metabolismo , Microtúbulos/metabolismo , Proteínas de Transporte/metabolismo
14.
Biochemistry ; 51(33): 6499-510, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22846040

RESUMO

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-L-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 Å resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.


Assuntos
Benzopiranos/farmacologia , Monossacarídeos/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Manosídeos/farmacologia , Modelos Moleculares , Proantocianidinas/farmacologia , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo
15.
J Biol Chem ; 286(40): 35163-75, 2011 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-21816819

RESUMO

PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Humanos , Luz , Modelos Estatísticos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Fatores de Troca de Nucleotídeo Guanina Rho , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Raios Ultravioleta , Raios X , Proteína rhoA de Ligação ao GTP/química
16.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 4): 243-8, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21460442

RESUMO

Macromolecular crystallography relies on the availability and quality of single crystals; these are typically obtained through extensive screening, which has a very low intrinsic success rate. Crystallization is not a completely stochastic process and many proteins do not succumb to crystallization because of specific microscopic features of their molecular surfaces. It follows that rational surface engineering through site-directed mutagenesis should allow a systematic and significant improvement in crystallization success rates. Here, one such established strategy, surface-entropy reduction (SER), is discussed, including its successes, limitations and possible future developments.


Assuntos
Cristalografia por Raios X/métodos , Proteínas/análise , Entropia , Escherichia coli/química , Modelos Moleculares , Mutação , Estrutura Quaternária de Proteína , Proteínas/química , Proteínas/genética , Propriedades de Superfície
17.
Acta Crystallogr A Found Adv ; 77(Pt 5): 362-378, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34473092

RESUMO

The bent structure of the water molecule, and its hydrogen-bonding properties, arguably rank among the most impactful discoveries in the history of chemistry. Although the fact that the H-O-H angle must deviate from linearity was inferred early in the 20th century, notably from the existence of the electric dipole moment, it was not clear what that angle should be and why. One hundred years ago, a young PhD student at the University of California, Berkeley, Eustace J. Cuy, rationalized the V-shape structure of a water molecule using the Lewis theory of a chemical bond, i.e. a shared electron pair, and its tetrahedral stereochemistry. He was inspired, in part, by the proposal of a weak (hydrogen) bond in water by two colleagues at Berkeley, Wendell Latimer and Worth Rodebush, who published their classic paper a year earlier. Cuy went on to suggest that other molecules, notably H2S and NH3, have similar structures, and presciently predicted that this architecture has broader consequences for the structure of water as a liquid. This short, but brilliant paper has been completely forgotten, perhaps due to the tragic death of the author at the age of 28; the hydrogen-bond study is also rarely recognized. One of the most impactful publications on the structure of liquid water, a classic treatise published in 1933 by John Bernal and Ralph Fowler, does not mention either of the two pioneering papers. In this essay, the background for the two discoveries is described, including the brief history of Lewis's research on the nature of the chemical bond, and the history of the discovery of the hydrogen bond, which inspired Cuy to look at the structure of the water molecule. This is - to the best of the author's knowledge - the first biographical sketch of Eustace J. Cuy.

18.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 5): 604-15, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20445236

RESUMO

Until recently, protein crystallization has mostly been regarded as a stochastic event over which the investigator has little or no control. With the dramatic technological advances in synchrotron-radiation sources and detectors and the equally impressive progress in crystallographic software, including automated model building and validation, crystallization has increasingly become the rate-limiting step in X-ray diffraction studies of macromolecules. However, with the advent of recombinant methods it has also become possible to engineer target proteins and their complexes for higher propensity to form crystals with desirable X-ray diffraction qualities. As most proteins that are under investigation today are obtained by heterologous overexpression, these techniques hold the promise of becoming routine tools with the potential to transform classical crystallization screening into a more rational high-success-rate approach. This article presents an overview of protein-engineering methods designed to enhance crystallizability and discusses a number of examples of their successful application.


Assuntos
Cristalografia por Raios X/métodos , Engenharia de Proteínas/métodos , Proteínas/química , Animais , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/química , Escherichia coli/química , Escherichia coli/genética , Proteínas Ligantes de Maltose , Modelos Moleculares , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Solubilidade
19.
Artigo em Inglês | MEDLINE | ID: mdl-20208147

RESUMO

The crystal structure of the dinB gene product from Geobacillus stearothermophilus (GsDinB) is reported at 2.5 A resolution. The dinB gene is one of the DNA-damage-induced genes and the corresponding protein, DinB, is the founding member of a Pfam family with no known function. The protein contains a four-helix up-down-down-up bundle that has previously been described in the literature in three disparate proteins: the enzyme MDMPI (mycothiol-dependent maleylpyruvate isomerase), YfiT and TTHA0303, a member of a small DUF (domain of unknown function). However, a search of the DALI structural database revealed similarities to a further 11 new unpublished structures contributed by structural genomics centers. The sequences of these proteins are quite divergent and represent several Pfam families, yet their structures are quite similar and most (but not all) seem to have the ability to coordinate a metal ion using a conserved histidine-triad motif. The structural similarities of these diverse proteins suggest that a new Pfam clan encompassing the families that share this fold should be created. The proteins that share this fold exhibit four different quaternary structures: monomeric and three different dimeric forms.


Assuntos
DNA Polimerase Dirigida por DNA/química , Geobacillus stearothermophilus/enzimologia , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína
20.
Biochemistry ; 48(36): 8664-71, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19653655

RESUMO

The DUF1094 family contains over 100 bacterial proteins, all containing a conserved CXC motif, with unknown function. We solved the crystal structure of the Bacillus subtilis representative, the product of the yphP gene. The protein shows remarkable structural similarity to thioredoxins, with a canonical alphabetaalphabetaalphabetabetaalpha topology, despite low amino acid sequence identity to thioredoxin. The CXC motif is found in the loop immediately downstream of the first beta-strand, in a location equivalent to the CXXC motif of thioredoxins, with the first Cys occupying a position equivalent to the first Cys in canonical thioredoxin. The experimentally determined reduction potential of YphP is E degrees' = -130 mV, significantly higher than that of thioredoxin and consistent with disulfide isomerase activity. Functional assays confirmed that the protein displays a level of isomerase activity that might be biologically significant. We propose a mechanism by which the members of this family catalyze isomerization using the CXC catalytic site.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Domínio Catalítico/fisiologia , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/fisiologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico/genética , Sequência Conservada/genética , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Isomerismo , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Isomerases de Dissulfetos de Proteínas/genética , Alinhamento de Sequência , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Tiorredoxinas/fisiologia
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