RESUMEN
Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies.
Asunto(s)
Anticuerpos Monoclonales/química , Biología Computacional/métodos , Algoritmos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Células CHO , Simulación por Computador , Cricetulus , Humanos , Mutación , Conformación Proteica , Ingeniería de Proteínas/métodos , Relación Estructura-ActividadRESUMEN
The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.
Asunto(s)
Secuencia de Aminoácidos , Anticuerpos Monoclonales , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Humanos , Estabilidad ProteicaRESUMEN
p38 MAPK and MAPK-activated protein kinase 2 (MK2) are key components of signaling pathways leading to many cellular responses, notably the proinflammatory cytokine production. The physical association of p38alpha isoform and MK2 is believed to be physiologically important for this signaling. We report the 2.7-A resolution crystal structure of the unphosphorylated complex between p38alpha and MK2. These protein kinases bind "head-to-head," present their respective active sites on approximately the same side of the heterodimer, and form extensive intermolecular interactions. Among these interactions, the MK2 Ile-366-Ala-390, which includes the bipartite nuclear localization signal, binds to the p38alpha-docking region. This binding supports the involvement of noncatalytic regions to the tight binding of the MK2:p38alpha binary assembly. The MK2 residues 345-365, containing the nuclear export signal, block access to the p38alpha active site. Some regulatory phosphorylation regions of both protein kinases engage in multiple interactions with one another in this complex. This structure gives new insights into the regulation of the protein kinases p38alpha and MK2, aids in the better understanding of their known cellular and biochemical studies, and provides a basis for understanding other regulatory protein-protein interactions involving signal transduction proteins.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Clonación Molecular , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Ratones , Complejos Multiproteicos/química , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Difracción de Rayos X , Proteínas Quinasas p38 Activadas por Mitógenos/químicaRESUMEN
Toluene 4-monooxygenase (T4MO) is a four-component complex that catalyzes the regiospecific, NADH-dependent hydroxylation of toluene to yield p-cresol. The catalytic effector (T4moD) of this complex is a 102-residue protein devoid of metals or organic cofactors. It forms a complex with the diiron hydroxylase component (T4moH) that influences both the kinetics and regiospecificity of catalysis. Here, we report crystal structures for native T4moD and two engineered variants with either four (DeltaN4-) or 10 (DeltaN10-) residues removed from the N-terminal at 2.1-, 1.7-, and 1.9-A resolution, respectively. The crystal structures have C-alpha root-mean-squared differences of less than 0.8 A for the central core consisting of residues 11-98, showing that alterations of the N-terminal have little influence on the folded core of the protein. The central core has the same fold topology as observed in the NMR structures of T4moD, the methane monooxygenase effector protein (MmoB) from two methanotrophs, and the phenol hydroxylase effector protein (DmpM). However, the root-mean-squared differences between comparable C-alpha positions in the X-ray structures and the NMR structures vary from approximately 1.8 A to greater than 6 A. The X-ray structures exhibit an estimated overall coordinate error from 0.095 (0.094) A based on the R-value (R free) for the highest resolution DeltaN4-T4moD structure to 0.211 (0.196) A for the native T4moD structure. Catalytic studies of the DeltaN4-, DeltaN7-, and DeltaN10- variants of T4moD show statistically insignificant changes in k(cat), K(M), k(cat)/K(M), and K(I) relative to the native protein. Moreover, there was no significant change in the regiospecificity of toluene oxidation with any of the T4moD variants. The relative insensitivity to changes in the N-terminal region distinguishes T4moD from the MmoB homologues, which each require the approximately 33 residue N-terminal region for catalytic activity.
Asunto(s)
Dominio Catalítico , Complejos Multienzimáticos/química , Complejos Multienzimáticos/fisiología , Oxigenasas/química , Oxigenasas/fisiología , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico/genética , Cristalización , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Resonancia Magnética Nuclear Biomolecular , Oxigenasas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Estructura Secundaria de Proteína/genética , Pseudomonas mendocina/enzimología , Pseudomonas mendocina/genética , Eliminación de Secuencia/genética , Relación Estructura-ActividadRESUMEN
Diffraction-quality crystals have been obtained of the xenobiotic reductase A (XenA) from Pseudomonas II-B, which was originally cultured from the contaminated soil of a World War II era munitions-manufacturing plant. Several complete X-ray diffraction data sets have been collected and analyzed. The native XenA data set includes reflections between 35 and 1.65 A. Four-wavelength MAD data sets from selenomethionine-enriched XenA and from three different ligand complexes are also reported. The XenA crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 84, b = 158, c = 57 A. Experimental phasing from analysis of the MAD data from selenomethionine-enriched XenA reveals the presence of two molecules in the asymmetric unit. They are related by a non-crystallographic 2(1) screw axis nearly parallel to the c axis, but offset by a quarter unit-cell translation. Thus, the local symmetry produces approximate systematic absences along the (00l) principal axis and complicates the space-group determination.
Asunto(s)
Proteínas Bacterianas/química , Flavoproteínas/química , Oxidorreductasas/química , Pseudomonas putida/enzimología , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Flavoproteínas/metabolismo , Ligandos , Metronidazol/química , Metronidazol/metabolismo , Nitroglicerina/química , Nitroglicerina/metabolismo , Oxidorreductasas/metabolismo , Conformación Proteica , Reproducibilidad de los Resultados , Selenometionina/química , Selenometionina/metabolismo , Trinitrotolueno/química , Trinitrotolueno/metabolismoRESUMEN
Single crystals have been obtained of the toluene 4-monooxygenase catalytic effector protein, the SeMet-enriched protein and a truncated isoform missing ten amino acids from the N-terminus. Complete X-ray diffraction data sets have been collected and analyzed to 2.0, 3.0 and 1.96 A resolution for the native, SeMet and truncated isoform crystals, respectively. The native and SeMet proteins crystallized in space group P6(1)22 (unit-cell parameters a = b = 86.41 +/- 0.15, c = 143.90 +/- 0.27 A), whereas the truncated isoform crystallized in space group P2(1)3 (a = b = c = 86.70 +/- 0.47 A). Matthews coefficient calculations suggest either two or three molecules per asymmetric unit in the P6(1)22 space group and two molecules per asymmetric unit in the P2(1)3 space group. Experimental phases from MAD analysis of the SeMet isoform and molecular replacement of the truncated isoform confirm the presence of two molecules per asymmetric unit in each case. These crystallographic results are the first available for the evolutionarily related but functionally diversified catalytic effector proteins from the multicomponent diiron monooxygenase family.
Asunto(s)
Oxigenasas/química , Cristalización , Cristalografía por Rayos X , Escherichia coli/metabolismo , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Oxigenasas/aislamiento & purificación , Pseudomonas/enzimologíaRESUMEN
The M3 protein encoded by murine gamma herpesvirus68 (gamma HV68) functions as an immune system saboteur by the engagement of chemoattractant cytokines, thereby altering host antiviral inflammatory responses. Here we report the crystal structures of M3 both alone and in complex with the CC chemokine MCP-1. M3 is a two-domain beta sandwich protein with a unique sequence and topology, forming a tightly packed anti-parallel dimer. The stoichiometry of the MCP-1:M3 complex is 2:2, with two monomeric chemokines embedded at distal ends of the preassociated M3 dimer. Conformational flexibility and electrostatic complementation are both used by M3 to achieve high-affinity and broad-spectrum chemokine engagement. M3 also employs structural mimicry to promiscuously sequester chemokines, engaging conservative structural elements associated with both chemokine homodimerization and binding to G protein-coupled receptors.
Asunto(s)
Quimiocina CCL2/química , Receptores Virales/química , Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dimerización , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , SolucionesRESUMEN
Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity for para hydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD-T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD-T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity with p-cresol representing >96% of total products from toluene. Similar coupling and regiospecificity for para hydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an approximately 20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so that p-cresol represents approximately 60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absence or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparent V(max)/K(M) specificities differing by approximately 2-4-fold and coupling efficiencies ranging from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity for ortho hydroxylation with all substrates tested except nitrobenzene, which gives only m-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.
Asunto(s)
Isoenzimas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas/metabolismo , Tolueno/metabolismo , Sitios de Unión , Catálisis , Transporte de Electrón , Conformación Molecular , Mutagénesis , Oxigenasas/genética , Unión Proteica , Pseudomonas/enzimología , Especificidad por SustratoRESUMEN
The expression of a gene, designated as Retroviral insertion site (Ris)2, was activated by retroviral DNA integration in an immortalized primitive erythroid cell line, EB-PE. Ris2 was also expressed at high levels in all human tumor cell lines analysed. Consistently, NIH3T3 fibroblasts overexpressing Ris2 formed tumors in Rag2 -/- mice when injected subcutaneously. The putative RIS2 protein shows a high sequence similarity to Xenopus CDT1, Drosophila DUP, and human CDT1, a newly identified DNA replication licensing protein, suggesting that Ris2 is a mouse homologue of CDT1. Cells overexpressing Ris2/Cdt1 exhibited a quicker entry into S phase when released from serum starvation compared to controls. Our results suggest that CDT1, an essential licensing protein for DNA replication, can function as an oncogene in mammals.