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1.
Cell ; 187(7): 1666-1684.e26, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38490194

RESUMO

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.


Assuntos
Inibidores Enzimáticos , Falência Hepática , MAP Quinase Quinase 4 , Animais , Humanos , Camundongos , Hepatectomia/métodos , Hepatócitos , Fígado , Hepatopatias/tratamento farmacológico , Falência Hepática/tratamento farmacológico , Falência Hepática/prevenção & controle , Regeneração Hepática , Suínos , MAP Quinase Quinase 4/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico
2.
Biochem J ; 477(22): 4383-4395, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33111951

RESUMO

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme-substrate complex.


Assuntos
Ativadores de Enzimas/química , Proteínas Fúngicas/química , Hypocreales/química , beta-Glucosidase/química , Ressonância Magnética Nuclear Biomolecular
3.
J Biomol NMR ; 74(10-11): 521-529, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32901320

RESUMO

No matter the source of compounds, drug discovery campaigns focused directly on the target are entirely dependent on a consistent stream of reliable data that reports on how a putative ligand interacts with the protein of interest. The data will derive from many sources including enzyme assays and many types of biophysical binding assays such as TR-FRET, SPR, thermophoresis and many others. Each method has its strengths and weaknesses, but none is as information rich and broadly applicable as NMR. Here we provide a number of examples of the utility of NMR for enabling and providing ongoing support for the early pre-clinical phase of small molecule drug discovery efforts. The examples have been selected for their usefulness in a commercial setting, with full understanding of the need for speed, cost-effectiveness and ease of implementation.


Assuntos
Descoberta de Drogas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Cristalografia/métodos , Ensaios de Triagem em Larga Escala , Ligantes , Proteínas/isolamento & purificação , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas/química , Soluções/química
4.
Biomol NMR Assign ; 14(2): 269, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32654087

RESUMO

In the original publication of the article, the name of one of the authors is incorrect. The author's name is Eiso AB, but was modified to A. B. Eiso. The correct name is given in this Correction.

5.
Angew Chem Int Ed Engl ; 59(46): 20508-20514, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32533782

RESUMO

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.


Assuntos
Glicosídeo Hidrolases/metabolismo , Hidrólise , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica
6.
Int J Mol Sci ; 20(15)2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31357624

RESUMO

The divisome is a large protein complex that regulates bacterial cell division and therefore represents an attractive target for novel antibacterial drugs. In this study, we report on the ligandability of FtsQ, which is considered a key component of the divisome. For this, the soluble periplasmic domain of Escherichia coli FtsQ was immobilized and used to screen a library of 1501 low molecular weight (< 300 Da), synthetic compounds for those that interact with the protein. A primary screen was performed using target immobilized NMR screening (TINS) and yielded 72 hits. Subsequently, these hits were validated in an orthogonal assay. At first, we aimed to do this using surface plasmon resonance (SPR), but the lack of positive control hampered optimization of the experiment. Alternatively, a two-dimensional heteronuclear single quantum coherence (HSQC) NMR spectrum of FtsQ was obtained and used to validate these hits by chemical shift perturbation (CSP) experiments. This resulted in the identification of three fragments with weak affinity for the periplasmic domain of FtsQ, arguing that the ligandability of FtsQ is low. While this indicates that developing high affinity ligands for FtsQ is far from straightforward, the identified hit fragments can help to further interrogate FtsQ interactions.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Divisão Celular , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Relação Estrutura-Atividade
7.
Essays Biochem ; 61(5): 485-493, 2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-29118095

RESUMO

NMR spectroscopy is a powerful technique that can provide valuable structural information for drug discovery endeavors. Here, we discuss the strengths (and limitations) of NMR applications to structure-based drug discovery, highlighting the different levels of resolution and throughput obtainable. Additionally, the emerging field of paramagnetic NMR in drug discovery and recent developments in approaches to speed up and automate protein-observed NMR data collection and analysis are discussed.


Assuntos
Desenho de Fármacos , Drogas em Investigação/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , Descoberta de Drogas/métodos , Drogas em Investigação/síntese química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas/agonistas , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-Atividade
8.
J Am Chem Soc ; 139(28): 9523-9533, 2017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28691806

RESUMO

Methyl groups are powerful probes for the analysis of structure, dynamics and function of supramolecular assemblies, using both solution- and solid-state NMR. Widespread application of the methodology has been limited due to the challenges associated with assigning spectral resonances to specific locations within a biomolecule. Here, we present Methyl Assignment by Graph Matching (MAGMA), for the automatic assignment of methyl resonances. A graph matching protocol examines all possibilities for each resonance in order to determine an exact assignment that includes a complete description of any ambiguity. MAGMA gives 100% accuracy in confident assignments when tested against both synthetic data, and 9 cross-validated examples using both solution- and solid-state NMR data. We show that this remarkable accuracy enables a user to distinguish between alternative protein structures. In a drug discovery application on HSP90, we show the method can rapidly and efficiently distinguish between possible ligand binding modes. By providing an exact and robust solution to methyl resonance assignment, MAGMA can facilitate significantly accelerated studies of supramolecular machines using methyl-based NMR spectroscopy.


Assuntos
Automação , Proteínas de Choque Térmico HSP90/química , Ressonância Magnética Nuclear Biomolecular , Algoritmos , Proteínas de Choque Térmico HSP90/genética , Humanos , Substâncias Macromoleculares/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida
9.
J Biol Chem ; 287(10): 7146-58, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22130672

RESUMO

Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that the RING finger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control.


Assuntos
Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Cádmio/química , Cádmio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Ligação Proteica/fisiologia , Domínios RING Finger , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases , Ubiquitinação/fisiologia , Zinco/química , Zinco/metabolismo
10.
J Biomol Screen ; 15(8): 978-89, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20817886

RESUMO

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM K(D) is limited. Similarly, they find that SPR detection is most suited to ligands that bind with K(D) better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteínas Imobilizadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/isolamento & purificação , Ressonância de Plasmônio de Superfície/métodos , Técnicas Biossensoriais/métodos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacologia , Ligantes , Modelos Biológicos , Terapia de Alvo Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Bibliotecas de Moléculas Pequenas/análise
11.
J Am Chem Soc ; 132(39): 13765-75, 2010 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-20828131

RESUMO

We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the prion domain of the fungal prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218-289) in its fibrillar state forms a left-handed ß-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.


Assuntos
Proteínas Fúngicas/química , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
12.
Chem Biol ; 17(8): 881-91, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20797617

RESUMO

Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here, we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The Escherichia coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1071 drug fragments for binding using target immobilized NMR Screening. Biochemical and biophysical validation of the eight most potent hits revealed an IC(50) range of 7-200 microM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Descoberta de Drogas/métodos , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/química , Escherichia coli , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Micelas , Nanotecnologia , Ligação Proteica , Estabilidade Proteica , Reprodutibilidade dos Testes , Solubilidade
13.
J Biol Chem ; 285(13): 10087-10097, 2010 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-20081198

RESUMO

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5'-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra alpha-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5'-phosphate recognition by the BRCT domains of bacterial NAD(+)-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions.


Assuntos
Proteína BRCA1/química , DNA/química , Proteína de Replicação C/química , Algoritmos , Sequência de Aminoácidos , Biologia Computacional/métodos , Replicação do DNA , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
14.
J Mol Biol ; 383(5): 1156-70, 2008 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-18804481

RESUMO

The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer alpha-beta sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , DNA/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Sequência Conservada , Enzimas Reparadoras do DNA , Endonucleases/metabolismo , Células Eucarióticas/química , Evolução Molecular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Células Procarióticas/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Soluções , Homologia Estrutural de Proteína
15.
Structure ; 16(1): 149-59, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18184592

RESUMO

The yeast Paf1 complex consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and regulates histone H2B ubiquitination, histone H3 methylation, RNA polymerase II carboxy-terminal domain (CTD) Ser2 phosphorylation, and RNA 3' end processing. We provide structural insight into the Paf1 complex with the NMR structure of the conserved and functionally important Plus3 domain of human Rtf1. A predominantly beta-stranded subdomain displays structural similarity to Dicer/Argonaute PAZ domains and to Tudor domains. We further demonstrate that the highly basic Rtf1 Plus3 domain can interact in vitro with single-stranded DNA via residues on the rim of the beta sheet, reminiscent of siRNA binding by PAZ domains, but did not detect binding to double-stranded DNA or RNA. We discuss the potential role of Rtf1 Plus3 ssDNA binding during transcription elongation.


Assuntos
Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Conformação Proteica , Alinhamento de Sequência , Fatores Supressores Imunológicos/genética , Transcrição Gênica
16.
Drug Discov Today ; 12(23-24): 1032-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18061882

RESUMO

With more than 10 years of practical experience and theoretical analysis, fragment-based drug discovery (FBDD) has entered the mainstream of the pharmaceutical and biotech industries. An array of biophysical techniques has been used to detect the weak interaction between a fragment and the target. Each technique presents its own requirements regarding the fragment collection and the target; therefore, in order to optimize the potential of FBDD, the nature of the target should be a driving factor for simultaneous development of both the library and the screening technology. A roadmap is now available to guide fragment-to-lead evolution when structural information is available. The next challenge is to apply FBDD to targets for which high-resolution structural information is not available.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Preparações Farmacêuticas/química , Bibliotecas de Moléculas Pequenas , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/tendências , Farmacologia , Relação Estrutura-Atividade
17.
J Biomol NMR ; 39(4): 331-6, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17922258

RESUMO

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of 1HN, 15N, 1Halpha, 13Calpha, 13CO and 13Cbeta chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas do Citoesqueleto/química , Ressonância Magnética Nuclear Biomolecular , Domínios de Homologia de src , Humanos , Conformação Proteica
18.
BMC Struct Biol ; 7: 22, 2007 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-17407569

RESUMO

BACKGROUND: SH3 domains are small protein modules of 60-85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the alpha-spectrin SH3 domain (Spc-SH3). RESULTS: Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex. CONCLUSION: Based on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding.


Assuntos
Alanina/genética , Arginina/genética , Proteínas Mutantes/química , Proteínas Proto-Oncogênicas c-abl/química , Espectrina/química , Espectrina/metabolismo , Domínios de Homologia de src , Animais , Varredura Diferencial de Calorimetria , Galinhas , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/metabolismo , Termodinâmica , Água
19.
J Biol Chem ; 281(49): 37993-8003, 2006 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-17001075

RESUMO

Spore formation is an extreme response of many bacterial species to starvation. In the case of pathogenic species of Bacillus and Clostridium, it is also a component of disease transmission. Entry into the pathway of sporulation in Bacillus subtilis and its relatives is controlled by an expanded two-component system in which starvation signals lead to the activation of sensor kinases and phosphorylation of the master sporulation response regulator Spo0A. Accumulation of threshold concentrations of Spo0A approximately P heralds the commitment to sporulation. Countering the activities of the sensor kinases are phosphatases such as Spo0E, which dephosphorylate Spo0A approximately P and inhibit sporulation. Spo0E-like protein-aspartic acid-phosphate phosphatases, consisting of 50-90 residues, are conserved in sporeforming bacteria and unrelated in sequence to proteins of known structure. Here we determined the structures of the Spo0A approximately P phosphatases BA1655 and BA5174 from Bacillus anthracis using nuclear magnetic resonance spectroscopy. Each is composed of two anti-parallel alpha-helices flanked by flexible regions at the termini. The signature SQELD motif (SRDLD in BA1655) is situated in the middle of helix alpha2 with its polar residues projecting outward. BA5174 is a monomer, whereas BA1655 is a dimer. The four-helix bundle structure in the dimer is reminiscent of the phosphotransferase Spo0B and the chemotaxis phosphatase CheZ, although in contrast to these systems, the subunits in BA1655 are in head-to-tail rather than head-to-head apposition. The implications of the structures for interactions between the phosphatases and their substrate Spo0A approximately P are discussed.


Assuntos
Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , Monoéster Fosfórico Hidrolases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacillus anthracis/genética , Bacillus anthracis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , DNA Bacteriano/genética , Dimerização , Genes Bacterianos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/fisiologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologia
20.
Biochemistry ; 45(35): 10606-13, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16939212

RESUMO

Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors. In this paper, we report on the solution structure of CSPsg4, a chemosensory protein from the desert locust Schistocerca gregaria, which is expressed in the antennae and other chemosensory organs. The 3D NMR structure revealed an overall fold consisting of six alpha-helices, spanning residues 13-18, 20-31, 40-54, 62-78, 80-90, and 97-103, connected by loops which in some cases show dihedral angles typical of beta-turns. As in the only other chemosensory protein whose structure has been solved so far, namely, CSP from the moth Mamestra brassicae, four helices are arranged to form a V-shaped motif; another helix runs across the two V's, and the last one is packed against the external face. Analysis of the tertiary structure evidenced multiple hydrophobic cavities which could be involved in ligand binding. In fact, incubation of the protein with a natural ligand, namely, oleamide, produced substantial changes to the NMR spectra, suggesting extensive conformational transitions upon ligand binding.


Assuntos
Gafanhotos/química , Proteínas de Insetos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
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