RESUMEN
P16(INK4A) (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly because of the prevalence of genetic inactivation of the p16(INK4A) (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that an elevated level of expression (upregulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications for the structure-function relationship of P16 and for human cancers.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Neoplasias/química , Neoplasias/terapia , Secuencia de Aminoácidos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Sistemas de Liberación de Medicamentos/métodos , Regulación Neoplásica de la Expresión Génica , Sitios Genéticos/genética , Humanos , Datos de Secuencia Molecular , Mutación , Neoplasias/genética , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
Phospholipase A2 catalyses the hydrolysis of the ester bond of 3-sn-phosphoglycerides. Here, we report the crystal structures of the free and anisic acid-bound triple mutant (K53,56,120M) of bovine pancreatic phospholipase A2. In the bound triple mutant structure, the small organic molecule p-anisic acid is found in the active site, and one of the carboxylate oxygen atoms is coordinated to the functionally important primary calcium ion. The other carboxylate oxygen atom is hydrogen bonded to the phenolic hydroxyl group of Tyr69. In addition, the bound anisic acid molecule replaces one of the functionally important water molecules in the active site. The residues 60-70, which are in a loop (surface loop), are disordered in most of the bovine pancreatic phospholipase A2 structures. It is interesting to note that these residues are ordered in the bound triple mutant structure but are disordered in the free triple mutant structure. The organic crystallization ingredient 2-methyl-2,4-pentanediol is found near the active site of the free triple mutant structure. The overall tertiary folding and stereochemical parameters for the final models of the free and anisic acid-bound triple mutant are virtually identical.
Asunto(s)
Hidroxibenzoatos/química , Modelos Moleculares , Fosfolipasas A/química , Sitio Alostérico , Animales , Sitios de Unión , Bovinos , Cristalización , Cristalografía por Rayos X , Éteres de Hidroxibenzoatos , Hidroxibenzoatos/metabolismo , Mutagénesis Sitio-Dirigida , Páncreas/enzimología , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Conformación ProteicaRESUMEN
This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H(epsilon 2) of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [15N(epsilon 2)]His. Instead, the downfield resonance arises from H(delta1) of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(+/-0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The pK(a) of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp em leader His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol).
Asunto(s)
Inhibidores Enzimáticos/química , Histidina/química , Fosfolipasas A/química , Animales , Ácido Aspártico/química , Ácido Aspártico/genética , Venenos de Abeja/química , Venenos de Abeja/enzimología , Sitios de Unión , Bovinos , Inhibidores Enzimáticos/metabolismo , Histidina/genética , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Fosfolipasas A/genética , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Fosfolípidos/química , ProtonesRESUMEN
Phospholipase A(2) catalyses hydrolysis of the ester bond at the C2 position of 3-sn-phosphoglycerides. Here we report the 1.9A resolution crystal structure of the triple mutant K56,120,121M of bovine pancreatic phospholipase A(2). The structure was solved by molecular replacement method using the orthorhombic form of the recombinant phospholipase A(2). The final protein model contains all the 123 amino acid residues, two calcium ions, 125 water molecules and one 2-methyl-2-4-pentanediol molecule. The model has been refined to a crystallographic R-factor of 19.6% (R(free) of 25.9%) for all data between 14.2A and 1.9A. The residues 62-66, which are in a surface loop, are always disordered in the structures of bovine pancreatic phospholipase A(2) and its mutants. It is interesting to note that the residues 62-66 in the present structure is ordered and the conformation varies substantially from those in the previously published structures of this enzyme. An unexpected and interesting observation in the present structure is that, in addition to the functionally important calcium ion in the active site, one more calcium ion is found near the N terminus. Detailed structural analyses suggest that binding of the second calcium ion could be responsible for the conformational change and the ordering of the surface loop. Furthermore, the results suggest a structural reciprocity between the k(cat)(*) allosteric site and surface loop at the i-face, which represents a newly identified structural property of secreted phospholipase A(2).
Asunto(s)
Calcio/química , Modelos Moleculares , Páncreas/enzimología , Fosfolipasas A/química , Fosfolipasas A/genética , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Bovinos , Cristalografía por Rayos X , Escherichia coli , Glicoles/química , Hidrólisis , Cuerpos de Inclusión/química , Mutagénesis Sitio-Dirigida , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Porcinos , Agua/químicaRESUMEN
Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.