RESUMEN
The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively ß-barrels composed of between 8 and 26 ß-strands. To explore the relationship between ß-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 ß-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the ß-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in ß-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in ß-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Glicina/química , Simulación de Dinámica Molecular , Conformación Proteica en Lámina beta , Dominios ProteicosRESUMEN
The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to â¼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane-protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.
Asunto(s)
Membrana Celular/química , Hidrógeno/química , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Difusión , Glicoforinas/química , Humanos , Movimiento (Física) , Péptidos/química , Fosfatidilcolinas/química , Multimerización de Proteína , Receptores Acoplados a Proteínas G/química , TermodinámicaRESUMEN
BtuB is a TonB-dependent transporter that permits the high-affinity binding and transport of cobalamin (CBL), or vitamin B12, across the asymmetric outer membrane (OM) of Gram-negative bacteria. It has been shown that Ca2+ binding is necessary for high-affinity binding of CBL to BtuB, and earlier simulations suggested that calcium ions serve to stabilize key substrate-binding extracellular loops. However, those simulations did not account for the lipopolysaccharides in the OM. To illuminate the roles of both Ca2+ and lipopolysaccharides in protein functionality, we performed simulations of apo and Ca2+-loaded BtuB in symmetric and asymmetric bilayers. The simulations reveal that the oligosaccharides of LPS stabilize the extracellular loops to some degree, apparently obviating the need for Ca2+. However, it is shown that Ca2+ ions stabilize a key substrate-binding loop to an even greater degree, as well as reposition specific CBL-binding residues, bringing them closer to the organization found in the CBL-bound structure. These results indicate the importance of including realistic membrane models when simulating outer-membrane proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Calcio/química , Calcio/metabolismo , Cationes Bivalentes/química , Cationes Bivalentes/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Simulación de Dinámica Molecular , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de Proteína , Agua/químicaRESUMEN
Gram-negative bacteria are distinguished in part by a second, outer membrane surrounding them. This membrane is distinct from others, possessing an outer leaflet composed not of typical phospholipids but rather large, highly charged molecules known as lipopolysaccharides. Therefore, modeling the structure and dynamics of proteins embedded in the outer membrane requires careful consideration of their native environment. In this review, we examine how simulations of such outer-membrane proteins have evolved over the last two decades, culminating most recently in detailed, highly accurate atomistic models of the outer membrane. We also draw attention to how the simulations have coupled with experiments to produce novel insights unattainable through a single approach. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Membrana Celular/química , Membrana Celular/ultraestructura , Bacterias Gramnegativas/química , Modelos Químicos , Sitios de Unión , Bacterias Gramnegativas/ultraestructura , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Unión Proteica , Conformación ProteicaRESUMEN
The outer membrane of Gram-negative bacteria is replete with a host of ß-barrel outer membrane proteins (OMPs). Despite serving a variety of essential functions, including immune response evasion, the exact mechanism of OMP folding and membrane insertion remains largely unclear. The ß-barrel assembly machinery complex is required for OMP biogenesis. Crystal structures and molecular dynamics (MD) simulations of the central and essential component, BamA, suggest a mechanism involving lateral opening of its barrel domain. MD simulations reported here reveal an additional feature of BamA: a substrate exit pore positioned above the lateral opening site. Disulfide crosslinks that prevent lateral opening and exit pore formation result in a loss of BamA function, which can be fully rescued by the reductant tris(2-carboxyethyl)phosphine. These data provide strong evidence that lateral opening and exit pore formation are required for BamA function.