RESUMO
Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Poliubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia , Enzimas Desubiquitinantes/genética , Ativação Enzimática , Humanos , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Ubiquitina Tiolesterase/genética , UbiquitinaçãoRESUMO
A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22-35 molecules per micelle. The addition of MA at concentrations up to 100 µg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 µg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation.
Assuntos
Ácidos Graxos não Esterificados , Micelas , Polissorbatos , Espalhamento a Baixo Ângulo , Polissorbatos/química , Ácidos Graxos não Esterificados/química , Ácido Mirístico/química , Composição de MedicamentosRESUMO
Small-angle X-ray scattering (SAXS) offers a way to examine the overall shape and oligomerization state of biological macromolecules under quasi native conditions in solution. In the past decades, SAXS has become a standard tool for structure biologists due to the availability of high brilliance X-ray sources and the development of data analysis/interpretation methods. Sample handling robots and software pipelines have significantly reduced the time necessary to conduct SAXS experiments. Presently, most synchrotrons feature beamlines dedicated to biological SAXS, and the SAXS-derived models are deposited into dedicated and accessible databases. The size of macromolecules that may be analyzed ranges from small peptides or snippets of nucleic acids to gigadalton large complexes or even entire viruses. Compared to other structural methods, sample preparation is straightforward, and the risk of inducing preparation artefacts is minimal. Very importantly, SAXS is a method of choice to study flexible systems like unfolded or disordered proteins, providing the structural ensembles compatible with the data. Although it may be utilized stand-alone, SAXS profits a lot from available experimental or predicted high-resolution data and information from complementary biophysical methods. Here, we show the basic principles of SAXS and review latest developments in the fields of hybrid modeling and flexible systems.
Assuntos
Substâncias Macromoleculares/química , Difração de Raios X/métodos , Biologia Computacional/métodos , Simulação por Computador , Modelos Moleculares , Conformação Molecular , Espalhamento a Baixo Ângulo , SoftwareRESUMO
Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.