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1.
J Med Chem ; 65(17): 11776-11787, 2022 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-35993839

RESUMEN

Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.


Asunto(s)
Quinolinas , Trypanosoma , Tripanosomiasis Africana , Animales , Microscopía por Crioelectrón , Modelos Animales de Enfermedad , Humanos , Ratones , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Quinolinas/farmacología , Quinolinas/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología
2.
Proc Natl Acad Sci U S A ; 107(46): 19850-5, 2010 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-21045134

RESUMEN

Coiled coils are extensively and successfully used nowadays to rationally design multistranded structures for applications, including basic research, biotechnology, nanotechnology, materials science, and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants switch the peptide's topology from a dimer to a trimer only when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can coexist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils. They therefore should have major implications for the rational design of coiled coils and consequently many applications using these popular oligomerization domains.


Asunto(s)
Modelos Moleculares , Estructura Cuaternaria de Proteína , Proteínas/química , Factor de Transcripción Activador 1/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Dicroismo Circular , Cristalografía por Rayos X , Luz , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Proteínas Mutantes/química , Multimerización de Proteína , Proteínas Protozoarias/química , Proteínas de Saccharomyces cerevisiae/química , Dispersión de Radiación , Ultracentrifugación
3.
Cell ; 138(2): 366-76, 2009 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-19632184

RESUMEN

Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.


Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Microtúbulos/química , Señales de Clasificación de Proteína , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Alineación de Secuencia
4.
J Biol Chem ; 284(23): 15640-9, 2009 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-19359244

RESUMEN

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.


Asunto(s)
Microtúbulos/fisiología , Estatmina/farmacología , Alanina/metabolismo , Animales , Axonema/efectos de los fármacos , Axonema/fisiología , Cinética , Microscopía por Video , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Fosforilación , Fosfoserina/metabolismo , Desnaturalización Proteica , Renaturación de Proteína , Erizos de Mar , Estatmina/metabolismo , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismo
5.
J Cell Biol ; 184(5): 691-706, 2009 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-19255245

RESUMEN

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.


Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Células CHO , Diferenciación Celular/fisiología , Cricetinae , Cricetulus , Dimerización , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/ultraestructura , Multimerización de Proteína , Estructura Terciaria de Proteína
6.
Curr Biol ; 18(3): 177-82, 2008 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-18249114

RESUMEN

Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.


Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Calcio/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Molécula de Interacción Estromal 1
7.
Biochemistry ; 47(2): 779-86, 2008 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-18081319

RESUMEN

The EB1+TIP protein family and its binding partners track growing plus ends of microtubules in cells and are thought to regulate their dynamics. Here we determined the effects of EB1 and the N-terminal CAP-Gly domain (p150n) of one of its major binding partners, p150Glued, both separately and together, on the dynamic instability parameters at plus ends of purified steady-state microtubules. With EB1 alone, the shortening rate, the extent of shortening, and the catastrophe frequency were suppressed in the absence of significant effects on the growth rate or rescue frequency. The effects of EB1 on dynamics were significantly different when p150n was added together with EB1. The rate and extent of shortening and the catastrophe frequency were suppressed 3-4 times more strongly than with EB1 alone. In addition, the EB1-p150n complex increased the rescue frequency and the mean length the microtubules grew, parameters that were not significantly affected by EB1 alone. Similarly, deletion of EB1's C-terminal tail, which is a crucial binding region for p150n, significantly increased the ability of EB1 to suppress shortening dynamics. EB1 by itself bound along the length of the microtubules with 1 mol of EB1 dimer bound per approximately 12 mol of tubulin dimer. Approximately twice the amount of EB1 was recruited to the microtubules in the presence of p150n. Our results indicate that inactivation of EB1's flexible C-terminal tail significantly changes EB1's ability to modulate microtubule dynamics. They further suggest that p150Glued may activate and thereby facilitate the recruitment of EB1 to the tips of microtubules to regulate their dynamics.


Asunto(s)
Polaridad Celular , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Bovinos , Complejo Dinactina , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Erizos de Mar , Relación Estructura-Actividad
8.
Nat Struct Mol Biol ; 14(10): 959-67, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17828277

RESUMEN

In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of p150(glued) and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly-mediated protein interactions. We demonstrate that the conserved GKNDG motif of CAP-Gly domains is responsible for targeting to the C-terminal EEY/F sequence motifs of CLIP170, EB proteins and microtubules. The CAP-Gly-EEY/F interaction is essential for the recruitment of the dynactin complex by CLIP170 and for activation of CLIP170. Our findings define the molecular basis of CAP-Gly domain function, including the tubulin detyrosination-tyrosination cycle. They further establish fundamental roles for the interaction between CAP-Gly proteins and C-terminal EEY/F sequence motifs in regulating complex and dynamic cellular processes.


Asunto(s)
Proteínas Asociadas a Microtúbulos , Proteínas de Neoplasias , Conformación Proteica , Relación Estructura-Actividad , Secuencia de Aminoácidos , Cristalografía por Rayos X , Complejo Dinactina , Dineínas/química , Dineínas/genética , Dineínas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia
9.
Proc Natl Acad Sci U S A ; 104(29): 12151-6, 2007 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-17616581

RESUMEN

The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG with the highly selective low-molecular-weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here, we report the 2.4 A x-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis.


Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/patogenicidad , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/química , Amidas/química , Amidas/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Tiofenos/química , Tiofenos/farmacología
10.
Proc Natl Acad Sci U S A ; 104(17): 7062-7, 2007 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-17438295

RESUMEN

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed "trigger sequence," that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation.


Asunto(s)
Proteínas de Unión al ADN/química , Leucina Zippers , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/química , Sustitución de Aminoácidos , Arginina , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas de Unión al ADN/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Proteínas Mutantes/química , Péptidos/química , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Soluciones , Termodinámica , Factores de Transcripción/metabolismo
11.
Mol Cell ; 23(5): 663-71, 2006 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-16949363

RESUMEN

Dynamic microtubule plus-end tracking protein (+TIP) networks are implicated in all functions of microtubules, but the molecular determinants of their interactions are largely unknown. Here, we have explored key binding modes of +TIPs by analyzing the interactions between selected CAP-Gly, EB-like, and carboxy-terminal EEY/F-COO(-) sequence motifs. X-ray crystallography and biophysical binding studies demonstrate that the beta2-beta3 loop of CAP-Gly domains determines EB-like motif binding specificity. They further show how CAP-Gly domains serve as recognition domains for EEY/F-COO(-) motifs, which represent characteristic and functionally important sequence elements in EB, CLIP-170, and alpha-tubulin. Our findings provide a molecular basis for understanding the modular interaction modes between alpha-tubulin, CLIPs, EB proteins, and the dynactin-dynein motor complex and suggest that multiple low-affinity binding sites in different combinations control dynamic +TIP networks at microtubule ends. They further offer insights into the structural consequences of genetic CAP-Gly domain defects found in severe human disorders.


Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
12.
J Biol Chem ; 281(23): 16078-83, 2006 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-16554300

RESUMEN

Stathmin is an intrinsically disordered protein implicated in the regulation of microtubule dynamics and in the development of cancer. The microtubule destabilizing activity of stathmin is down-regulated by phosphorylation of four serine residues, Ser16, Ser25, Ser38, and Ser63. Here we have used calorimetric and spectroscopic methods, including nuclear magnetic resonance to analyze the properties of seven stathmin phosphoisoforms to bind tubulin and inhibit microtubule formation. We found that stathmin phosphorylation results in a substantial loss in hydration entropy upon tubulin-stathmin complex formation. Remarkably, a linear correlation between the free energy change of complex formation and the microtubule inhibition activities of stathmin phosphoisoforms was observed. This finding provides a biophysical basis for understanding the mechanism by which local stathmin activity gradients important for promoting localized microtubule growth are established. We further found that phosphorylation of Ser16 and Ser63 disrupts the formation of a tubulin-interacting beta-hairpin and a helical segment, respectively, explaining the dominant role of these residues in regulating cell cycle progression. The insight into the tubulin-stathmin interaction offers a molecular basis for understanding the nature and the factors that control intrinsically disordered protein systems in general.


Asunto(s)
Estatmina/metabolismo , Dicroismo Circular , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Termodinámica
13.
Proc Natl Acad Sci U S A ; 102(39): 13891-6, 2005 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-16172398

RESUMEN

In recent years, short coiled coils have been used for applications ranging from biomaterial to medical sciences. For many of these applications knowledge of the factors that control the topology of the engineered protein systems is essential. Here, we demonstrate that trimerization of short coiled coils is determined by a distinct structural motif that encompasses specific networks of surface salt bridges and optimal hydrophobic packing interactions. The motif is conserved among intracellular, extracellular, viral, and synthetic proteins and defines a universal molecular determinant for trimer formation of short coiled coils. In addition to being of particular interest for the biotechnological production of candidate therapeutic proteins, these findings may be of interest for viral drug development strategies.


Asunto(s)
Secuencias de Aminoácidos , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Ratones , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Proteínas/química
14.
EMBO J ; 24(2): 261-9, 2005 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-15616574

RESUMEN

EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. We find that EB1 is a stable dimer with a parallel coiled coil and show that dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 microM affinity. The conserved APC Ile2805-Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789-Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC-EB1 is post-translationally regulated in cells. Our findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Secuencia de Aminoácidos , Cristalografía , Proteínas de Escherichia coli/química , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Plásmidos , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido
15.
J Biol Chem ; 278(40): 38926-34, 2003 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-12860982

RESUMEN

Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degrees C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K0 = 6.0 x 10(6) m(-1). The obtained large negative molar heat capacity change of deltaCp0 = -860 cal mol(-1) K(-1) (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.


Asunto(s)
Proteínas de Microtúbulos , Fosfoproteínas/química , Tubulina (Proteína)/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calorimetría , Bovinos , Dicroismo Circular , ADN Complementario/metabolismo , Entropía , Glicerol/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Modelos Moleculares , Modelos Estadísticos , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Estatmina , Temperatura , Termodinámica , Factores de Tiempo , Tubulina (Proteína)/metabolismo
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