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1.
PLoS Biol ; 19(12): e3001474, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34879065

RESUMEN

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.


Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/genética , Proteínas de la Membrana/fisiología , Enzimas Ubiquitina-Conjugadoras/fisiología , Secuencia de Aminoácidos/genética , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Degradación Asociada con el Retículo Endoplásmico/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Unión Proteica/genética , Dominios Proteicos/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitinación
2.
J Biol Chem ; 295(9): 2664-2675, 2020 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-31974162

RESUMEN

Engineering and bioconjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multidomain protein may be complicated by undesirable side reactions to noninvolved domains. Furthermore, the advantages of segmental (or domain-specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods-expressed protein ligation, protein trans-splicing, and native chemical ligation-each have specific limitations. Here, we evaluated the use of different variants of Staphylococcus aureus sortase A for a range of ligation reactions and demonstrate that conditions can readily be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.


Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Ingeniería de Proteínas/métodos , Staphylococcus aureus/enzimología , Dominios Proteicos
3.
J Biomol NMR ; 74(4-5): 223-228, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32333192

RESUMEN

Recent methyl adiabatic relaxation dispersion experiments provide examination of conformational dynamics across a very wide timescale (102-105 s-1) and, particularly, provide insight into the hydrophobic core of proteins and allosteric effects associated with modulators. The experiments require efficient decoupling of 1H and 13C spin interactions, and some artifacts have been discovered, which are associated with the design of the proton decoupling scheme. The experimental data suggest that the original design is valid; however, pulse sequences with either no proton decoupling or proton decoupling with imperfect pulses can potentially exhibit complications in the experiments. Here, we demonstrate that pulse imperfections in the proton decoupling scheme can be dramatically alleviated by using a single composite π pulse and provide pure single-exponential relaxation data. It allows the opportunity to access high-quality methyl adiabatic relaxation dispersion data by removing the cross-correlation between dipole-dipole interaction and chemical shift anisotropy. The resulting high-quality data is illustrated with the binding of an allosteric modulator (G2BR) to the ubiquitin conjugating enzyme Ube2g2.


Asunto(s)
Artefactos , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Conformación Proteica , Receptores del Factor Autocrino de Motilidad/química , Enzimas Ubiquitina-Conjugadoras/química , Regulación Alostérica , Sitios de Unión , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Pliegue de Proteína , Protones , Receptores del Factor Autocrino de Motilidad/genética , Proteínas Recombinantes de Fusión/metabolismo , Termodinámica , Enzimas Ubiquitina-Conjugadoras/metabolismo
4.
Molecules ; 25(24)2020 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-33333809

RESUMEN

The ubiquitination pathway is central to many cell signaling and regulatory events. One of the intriguing aspects of the pathway is the combinatorial sophistication of substrate recognition and ubiquitin chain building determinations. The abundant structural and biological data portray several characteristic protein folds among E2 and E3 proteins, and the understanding of the combinatorial complexity that enables interaction with much of the human proteome is a major goal to developing targeted and selective manipulation of the pathway. With the commonality of some folds, there are likely other aspects that can provide differentiation and recognition. These aspects involve allosteric effects and conformational dynamics that can direct recognition and chain building processes. In this review, we will describe the current state of the knowledge for conformational dynamics across a wide timescale, address the limitations of present approaches, and illustrate the potential to make new advances in connecting dynamics with ubiquitination regulation.


Asunto(s)
Proteínas/química , Proteínas/metabolismo , Ubiquitinación , Humanos , Conformación Proteica , Pliegue de Proteína
5.
Biochim Biophys Acta ; 1860(1 Pt B): 325-32, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26459004

RESUMEN

BACKGROUND: The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin. METHODS: We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting with the solution-state NMR structures of γS-WT and γS-G18V. RESULTS: γS-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in γS-G18V that are not present in γS-WT. Molecular docking predicts three binding sites that are specific to γS-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in γS-WT. CONCLUSIONS: Although both proteins display non-specific binding, more residues are involved in ANS binding to γS-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site. GENERAL SIGNIFICANCE: Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.


Asunto(s)
Catarata/metabolismo , Simulación del Acoplamiento Molecular , Multimerización de Proteína , gamma-Cristalinas/química , gamma-Cristalinas/ultraestructura , Sitios de Unión , Catarata/genética , Variación Genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Conformación Proteica , Propiedades de Superficie , gamma-Cristalinas/genética
6.
J Am Chem Soc ; 138(16): 5392-402, 2016 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-27052457

RESUMEN

We demonstrate that the effect of protein crowding is critically dependent on the stability of the protein's hydration shell, which can dramatically vary between different proteins. In the human eye lens, γS-crystallin (γS-WT) forms a densely packed transparent hydrogel with a high refractive index, making it an ideal system for studying the effects of protein crowding. A single point mutation generates the cataract-related variant γS-G18V, dramatically altering the optical properties of the eye lens. This system offers an opportunity to explore fundamental questions regarding the effect of protein crowding, using γS-WT and γS-G18V: (i) how do the diffusion dynamics of hydration water change as a function of protein crowding?; and (ii) upon hydrogel formation of γS-WT, has a dynamic transition occurred generating a single population of hydration water, or do populations of bulk and hydration water coexist? Using localized spin probes, we separately probe the local translational diffusivity of both surface hydration and interstitial water of γS-WT and γS-G18V in solution. Surprisingly, we find that under the influence of hydrogel formation at highly crowded γS-WT concentrations up to 500 mg/mL, the protein hydration shell remains remarkably dynamic, slowing by less than a factor of 2, if at all, compared to that in dilute protein solutions of ∼5 mg/mL. Upon self-crowding, the population of this robust surface hydration water increases, while a significant bulk-like water population coexists even at ∼500 mg/mL protein concentrations. In contrast, surface water of γS-G18V irreversibly dehydrates with moderate concentration increases or subtle alterations to the solution conditions, demonstrating that the effect of protein crowding is highly dependent on the stability of the protein-specific hydration shell. The core function of γS-crystallin in the eye lens may be precisely its capacity to preserve a robust hydration shell, whose stability is abolished by a single G18V mutation.


Asunto(s)
gamma-Cristalinas/química , gamma-Cristalinas/genética , Amidas/química , Catarata/genética , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Hidrogeles/química , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Mutación , Estabilidad Proteica , Agua/química
7.
PLoS One ; 16(10): e0258429, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34648536

RESUMEN

Static light scattering is a popular physical chemistry technique that enables calculation of physical attributes such as the radius of gyration and the second virial coefficient for a macromolecule (e.g., a polymer or a protein) in solution. The second virial coefficient is a physical quantity that characterizes the magnitude and sign of pairwise interactions between particles, and hence is related to aggregation propensity, a property of considerable scientific and practical interest. Estimating the second virial coefficient from experimental data is challenging due both to the degree of precision required and the complexity of the error structure involved. In contrast to conventional approaches based on heuristic ordinary least squares estimates, Bayesian inference for the second virial coefficient allows explicit modeling of error processes, incorporation of prior information, and the ability to directly test competing physical models. Here, we introduce a fully Bayesian model for static light scattering experiments on small-particle systems, with joint inference for concentration, index of refraction, oligomer size, and the second virial coefficient. We apply our proposed model to study the aggregation behavior of hen egg-white lysozyme and human γS-crystallin using in-house experimental data. Based on these observations, we also perform a simulation study on the primary drivers of uncertainty in this family of experiments, showing in particular the potential for improved monitoring and control of concentration to aid inference.


Asunto(s)
Dispersión Dinámica de Luz , Muramidasa/química , gamma-Cristalinas/química , Animales , Teorema de Bayes , Pollos , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Muramidasa/metabolismo , Agregado de Proteínas , Cloruro de Sodio/química , gamma-Cristalinas/metabolismo
8.
J Phys Condens Matter ; 30(43): 435101, 2018 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-30280702

RESUMEN

The refractive index gradient of the eye lens is controlled by the concentration and distribution of its component crystallin proteins, which are highly enriched in polarizable amino acids. The current understanding of the refractive index increment ([Formula: see text]) of proteins is described using an additive model wherein the refractivity and specific volume of each amino acid type contributes according to abundance in the primary sequence. Here we present experimental measurements of [Formula: see text] for crystallins from the human lens and those of aquatic animals under uniform solvent conditions. In all cases, the measured values are much higher than those predicted from primary sequence alone, suggesting that structural factors also contribute to protein refractive index.


Asunto(s)
Cristalinas/química , Cristalinas/metabolismo , Refractometría , Animales , Humanos , Conformación Proteica
9.
J Phys Chem B ; 120(33): 8115-26, 2016 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-27063730

RESUMEN

We present a novel multi-conformation Monte Carlo simulation method that enables the modeling of protein-protein interactions and aggregation in crowded protein solutions. This approach is relevant to a molecular-scale description of realistic biological environments, including the cytoplasm and the extracellular matrix, which are characterized by high concentrations of biomolecular solutes (e.g., 300-400 mg/mL for proteins and nucleic acids in the cytoplasm of Escherichia coli). Simulation of such environments necessitates the inclusion of a large number of protein molecules. Therefore, computationally inexpensive methods, such as rigid-body Brownian dynamics (BD) or Monte Carlo simulations, can be particularly useful. However, as we demonstrate herein, the rigid-body representation typically employed in simulations of many-protein systems gives rise to certain artifacts in protein-protein interactions. Our approach allows us to incorporate molecular flexibility in Monte Carlo simulations at low computational cost, thereby eliminating ambiguities arising from structure selection in rigid-body simulations. We benchmark and validate the methodology using simulations of hen egg white lysozyme in solution, a well-studied system for which extensive experimental data, including osmotic second virial coefficients, small-angle scattering structure factors, and multiple structures determined by X-ray and neutron crystallography and solution NMR, as well as rigid-body BD simulation results, are available for comparison.


Asunto(s)
Citoplasma/química , Proteínas de Escherichia coli/química , Matriz Extracelular/química , Método de Montecarlo , Muramidasa/química , Animales , Artefactos , Benchmarking , Pollos , Cristalografía , Escherichia coli/química , Simulación de Dinámica Molecular , Conformación Proteica , Termodinámica
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