RESUMO
Computational and structure guided methods can make significant contributions to the development of solutions for difficult protein engineering problems, including the optimization of next generation of engineered antibodies. In this paper, we describe a contemporary industrial antibody engineering program, based on hypothesis-driven in silico protein optimization method. The foundational concepts and methods of computational protein engineering are discussed, and an example of a computational modeling and structure-guided protein engineering workflow is provided for the design of best-in-class heterodimeric Fc with high purity and favorable biophysical properties. We present the engineering rationale as well as structural and functional characterization data on these engineered designs.
Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Simulação de Dinâmica Molecular , Substituição de Aminoácidos , Animais , Especificidade de Anticorpos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/química , Engenharia de Proteínas , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Células CHO , Cromatografia Líquida , Cricetinae , Cricetulus , Desenho de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Espectrometria de Massas , Modelos Moleculares , Mutação , Engenharia de Proteínas/métodos , Multimerização Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , TemperaturaRESUMO
Assembly of the type-III secretion apparatus, which translocates proteins through both membranes of Gram-negative bacterial pathogens into host cells, requires the formation of an integral outer-membrane secretin ring. Typically, a small lipidated pilot protein is necessary for the stabilization and localization of this ring. Using NMR spectroscopy, we demonstrate that the C-terminal residues 553-570 of the Shigella flexneri secretin MxiD encompass the minimal binding domain for its cognate pilot MxiM. Although unstructured in isolation, upon complex formation with MxiM, these residues fold into an amphipathic turn-helix motif that caps the elongated hydrophobic cavity of the cracked beta-barrel pilot. Along with a rearrangement of core aromatic residues, this prevents the binding of lipids within the cavity. The mutually exclusive association of lipids and MxiD with MxiM establishes a framework for understanding the role of a pilot in the outer-membrane insertion and multimerization of the secretin ring.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Lipoproteínas/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação/efeitos dos fármacos , Detergentes/farmacologia , Dimerização , Lipoproteínas/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Shigella flexneri , Especificidade por SubstratoRESUMO
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Escherichia coli Enteropatogênica/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fatores de Virulência/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Escherichia coli Enteropatogênica/patogenicidade , Células HeLa , HumanosRESUMO
Hydrogen atoms are a vital component of enzyme structure and function. In recent years, atomic resolution crystallography (>or=1.2 A) has been successfully used to investigate the role of the hydrogen atom in enzymatic catalysis. Here, atomic resolution crystallography was used to study the effect of pH on cholesterol oxidase from Streptomyces sp., a flavoenzyme oxidoreductase. Crystallographic observations of the anionic oxidized flavin cofactor at basic pH are consistent with the UV-visible absorption profile of the enzyme and readily explain the reversible pH-dependent loss of oxidation activity. Furthermore, a hydrogen atom, positioned at an unusually short distance from the main chain carbonyl oxygen of Met122 at high pH, was observed, suggesting a previously unknown mechanism of cofactor stabilization. This study shows how a redox active site responds to changes in the enzyme's environment and how these changes are able to influence the mechanism of enzymatic catalysis.
Assuntos
Colesterol Oxidase/química , Flavoproteínas/química , Catálise , Colesterol Oxidase/metabolismo , Coenzimas/química , Coenzimas/metabolismo , Cristalografia por Raios X , Flavoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Químicos , Oxirredutases/química , Oxirredutases/metabolismo , Conformação Proteica , Streptomyces/enzimologia , Especificidade por Substrato/genéticaRESUMO
The ability to translocate virulence proteins into host cells through a type III secretion apparatus (TTSS) is a hallmark of several Gram-negative pathogens including Shigella, Salmonella, Yersinia, Pseudomonas, and enteropathogenic Escherichia coli. In common with other types of bacterial secretion apparatus, the assembly of the TTSS complex requires the preceding formation of its integral outer membrane secretin ring component. We have determined at 1.5 A the structure of MxiM28-142, the Shigella pilot protein that is essential for the assembly and membrane association of the Shigella secretin, MxiD. This represents the first atomic structure of a secretin pilot protein from the several bacterial secretion systems containing an orthologous secretin component. A deep hydrophobic cavity is observed in the novel 'cracked barrel' structure of MxiM, providing a specific binding domain for the acyl chains of bacterial lipids, a proposal that is supported by our various lipid/MxiM complex structures. Isothermal titration analysis shows that the C-terminal domain of the secretin, MxiD525-570, hinders lipid binding to MxiM.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/química , Shigella flexneri/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Cristalização , Lipoproteínas/fisiologia , Conformação ProteicaRESUMO
The X-ray crystal structure of the flavoenzyme cholesterol oxidase, SCOA (Streptomyces sp.SA-COO) has been determined to 0.95 A resolution. The large size (55kDa) and the high-resolution diffraction of this protein provides a unique opportunity to observe detailed electronic effects within a protein environment and to obtain a larger sampling for which to analyze these electronic and structural differences. It is well-known through spectroscopic methods that peptide carbonyl groups are polarized in alpha-helices. This electronic characteristic is evident in the sub-Angstrom electron density of SCOA. Our analysis indicates an increased tendency for the electron density of the main chain carbonyl groups within alpha-helices to be polarized toward the oxygen atoms. In contrast, the carbonyl groups in beta-sheet structures tend to exhibit a greater charge density between the carbon and oxygen atoms. Interestingly, the electronic differences observed at the carbonyl groups do not appear to be correlated to the bond distance of the peptide bond or the peptide planarity. This study gives important insight into the electronic effects of alpha-helix dipoles in enzymes and provides experimentally based observations that have not been previously characterized in protein structure.
Assuntos
Colesterol Oxidase/química , Proteínas de Bactérias/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Secundária de Proteína , Streptomyces/enzimologiaRESUMO
The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.