RESUMEN
The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.
Asunto(s)
Carboxipeptidasas , Proteínas Asociadas a Microtúbulos , Microtúbulos , Procesamiento Proteico-Postraduccional , Tubulina (Proteína) , Tirosina , Animales , Carboxipeptidasas/genética , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/química , Tubulina (Proteína)/química , Tirosina/químicaRESUMEN
The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Proteínas/química , Programas Informáticos , Secuencia de Aminoácidos , Biología Computacional , Microscopía por Crioelectrón , Cristalografía por Rayos X , Análisis de Secuencia de ProteínaRESUMEN
Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.
Asunto(s)
Cápside/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , ARN Viral/metabolismo , Rhinovirus/metabolismo , Activación Viral/fisiología , Desencapsidación Viral/fisiología , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Infecciones por Enterovirus/metabolismo , Infecciones por Enterovirus/virología , Genoma Viral/genética , Células HeLa , Humanos , Molécula 1 de Adhesión Intercelular/química , Molécula 1 de Adhesión Intercelular/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , ARN Viral/química , ARN Viral/genética , Rhinovirus/genética , Rhinovirus/fisiología , Homología de Secuencia de Aminoácido , Virión/genética , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
Asunto(s)
Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sitios de Unión , Dominios FERM , Humanos , Modelos Moleculares , Receptores de LDL/química , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
J-DNA-binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (ß-d-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozoa such as Trypanosoma and Leishmania JBP1 has two known functional domains: an N-terminal T hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins and a J-DNA-binding domain (JDBD) that resides in the middle of JBP1. Here, we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered structure. We found that this Δ-JDBD domain retains TH activity in vitro but displays a 15-fold lower apparent rate of hydroxylation compared with JBP1. Small-angle X-ray scattering (SAXS) experiments on JBP1 and JDBD in the presence or absence of J-DNA and on Δ-JDBD enabled us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate T hydroxylation 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module into the Δ-JDBD scaffold during evolution provided a mechanism that synergized J recognition and T hydroxylation, ensuring inheritance of base J in specific sequence patterns following DNA replication in kinetoplastid parasites.
Asunto(s)
ADN Protozoario/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Leishmania/química , Oxigenasas de Función Mixta/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma/química , Sitios de Unión , ADN Protozoario/química , Proteínas de Unión al ADN/genética , Leishmania/metabolismo , Oxigenasas de Función Mixta/química , Modelos Moleculares , Conformación Proteica , Proteínas Protozoarias/genética , Trypanosoma/metabolismoRESUMEN
The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica , Dominios Proteicos , Tubulina (Proteína)/metabolismoRESUMEN
J-base binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (ß-D-glucosylhydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single-domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance screening identified Nb6 as a strong binder, recognizing JBP1 with a 1:1 stoichiometry and high affinity (Kd = 30â nM). Crystallization trials of JBP1 in complex with Nb6 yielded crystals that diffracted to 1.47â Å resolution. However, the dimensions of the asymmetric unit and molecular replacement with a nanobody structure clearly showed that the crystals of the expected complex with JBP1 were of the nanobody alone. Nb6 crystallizes in space group P31 with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64â Å. Ensemble refinement suggests that in the ligand-free state one of the complementarity-determining regions (CDRs) is flexible, while the other two adopt well defined conformations.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Protozoarias/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Animales , Camélidos del Nuevo Mundo , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Glucósidos/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas Protozoarias/química , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Resonancia por Plasmón de Superficie , Uracilo/análogos & derivados , Uracilo/metabolismoRESUMEN
Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of âº-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of âº-tubulin, and purified vasohibins removed the C-terminal tyrosine of âº-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Carboxipeptidasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Proteínas Angiogénicas/genética , Biocatálisis , Carboxipeptidasas/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Haploidia , Humanos , Neovascularización FisiológicaRESUMEN
Cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules through kinetochores and the spindle assembly checkpoint (SAC) is satisfied. We show that the amino-terminal localization module of the SAC protein kinase MPS1 (monopolar spindle 1) directly interacts with the HEC1 (highly expressed in cancer 1) calponin homology domain in the NDC80 (nuclear division cycle 80) kinetochore complex in vitro, in a phosphorylation-dependent manner. Microtubule polymers disrupted this interaction. In cells, MPS1 binding to kinetochores or to ectopic NDC80 complexes was prevented by end-on microtubule attachment, independent of known kinetochore protein-removal mechanisms. Competition for kinetochore binding between SAC proteins and microtubules provides a direct and perhaps evolutionarily conserved way to detect a properly organized spindle ready for cell division.