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1.
Mol Cell ; 83(15): 2739-2752.e5, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37499662

RESUMO

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.


Assuntos
Lisofosfolipídeos , Esfingosina , Humanos , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo
2.
Nature ; 615(7952): 541-547, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36890228

RESUMO

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.


Assuntos
Atmosfera , Hidrogênio , Hidrogenase , Mycobacterium smegmatis , Microscopia Crioeletrônica , Hidrogênio/química , Hidrogênio/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Hidrogenase/ultraestrutura , Oxirredução , Oxigênio , Vitamina K 2/metabolismo , Atmosfera/química , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/metabolismo , Hidrogenação
3.
J Biol Chem ; 300(10): 107754, 2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39260694

RESUMO

The rise in multi-drug resistant Gram-negative bacterial infections has led to an increased need for "last-resort" antibiotics such as polymyxins. However, the emergence of polymyxin-resistant strains threatens to bring about a post-antibiotic era. Thus, there is a need to develop new polymyxin-based antibiotics, but a lack of knowledge of the mechanism of action of polymyxins hinders such efforts. It has recently been suggested that polymyxins induce cell lysis of the Gram-negative bacterial inner membrane (IM) by targeting trace amounts of lipopolysaccharide (LPS) localized there. We use multiscale molecular dynamics (MD), including long-timescale coarse-grained (CG) and all-atom (AA) simulations, to investigate the interactions of polymyxin B1 (PMB1) with bacterial IM models containing phospholipids (PLs), small quantities of LPS, and IM proteins. LPS was observed to (transiently) phase separate from PLs at multiple LPS concentrations, and associate with proteins in the IM. PMB1 spontaneously inserted into the IM and localized at the LPS-PL interface, where it cross-linked lipid headgroups via hydrogen bonds, sampling a wide range of interfacial environments. In the presence of membrane proteins, a small number of PMB1 molecules formed interactions with them, in a manner that was modulated by local LPS molecules. Electroporation-driven translocation of PMB1 via water-filled pores was favored at the protein-PL interface, supporting the 'destabilizing' role proteins may have within the IM. Overall, this in-depth characterization of PMB1 modes of interaction reveals how small amounts of mislocalized LPS may play a role in pre-lytic targeting and provides insights that may facilitate rational improvement of polymyxin-based antibiotics.

4.
Proc Natl Acad Sci U S A ; 119(42): e2211672119, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36215462

RESUMO

A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor-binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent secondary infection by the same or a closely related phage and nonproductive phage adsorption to lysed cell fragments, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a nonnative conformation of the plug of FhuA, causing the inward folding of two extracellular loops via "reverse" allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of Escherichia coli by phage T5 and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.


Assuntos
Bacteriófagos , Proteínas de Escherichia coli , Superinfecção , Proteínas da Membrana Bacteriana Externa/metabolismo , Receptores de Bacteriófagos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Lipoproteínas/metabolismo , Receptores Virais/metabolismo , Fagos T/química , Fagos T/metabolismo
5.
PLoS Comput Biol ; 19(1): e1010841, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36638139

RESUMO

The outer membrane factor CmeC of the efflux machinery CmeABC plays an important role in conferring antibiotic and bile resistance to Campylobacter jejuni. Curiously, the protein is N-glycosylated, with the glycans playing a key role in the effective function of this system. In this work we have employed atomistic equilibrium molecular dynamics simulations of CmeC in a representative model of the C. jejuni outer membrane to characterise the dynamics of the protein and its associated glycans. We show that the glycans are more conformationally labile than had previously been thought. The extracellular loops of CmeC visit the open and closed states freely suggesting the absence of a gating mechanism on this side, while the narrow periplasmic entrance remains tightly closed, regulated via coordination to solvated cations. We identify several cation binding sites on the interior surface of the protein. Additionally, we used steered molecular dynamics simulations to elucidate translocation pathways for a bile acid and a macrolide antibiotic. These, and additional equilibrium simulations suggest that the anionic bile acid utilises multivalent cations to climb the ladder of acidic residues that line the interior surface of the protein.


Assuntos
Campylobacter jejuni , Farmacorresistência Bacteriana Múltipla , Antibacterianos/farmacologia , Glicosilação , Ácidos e Sais Biliares/metabolismo , Proteínas de Bactérias/metabolismo
6.
Nat Methods ; 17(5): 505-508, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371966

RESUMO

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.


Assuntos
Lipídeos/análise , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Metaboloma , Proteoma/análise , Humanos , Ligantes
7.
Int J High Perform Comput Appl ; 37(1): 28-44, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36647365

RESUMO

We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus obscure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized.

8.
Microbiology (Reading) ; 168(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35294337

RESUMO

Bacterial cell envelopes are compositionally complex and crowded and while highly dynamic in some areas, their molecular motion is very limited, to the point of being almost static in others. Therefore, it is no real surprise that studying them at high resolution across a range of temporal and spatial scales requires a number of different techniques. Details at atomistic to molecular scales for up to tens of microseconds are now within range for molecular dynamics simulations. Here we review how such simulations have contributed to our current understanding of the cell envelopes of Gram-negative bacteria.


Assuntos
Parede Celular , Bactérias Gram-Negativas , Membrana Celular , Bactérias Gram-Negativas/genética , Simulação de Dinâmica Molecular
9.
Methods ; 185: 15-27, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32084518

RESUMO

Membrane proteins are amphipathic macromolecules whose exposed hydrophobic surfaces promote interactions with lipid membranes. Membrane proteins are remarkably diverse in terms of chemical composition and correspondingly, their biological functions and general biophysical behavior. Conventional experimental techniques provide an approach to study specific properties of membrane proteins e.g. their surface features, the nature and abundance of stabilizing intramolecular forces, preferred bilayer orientation, and the characteristics of their annular lipid shells. Molecular modeling software-and in particular, the suite of molecular dynamics algorithms-enables a more comprehensive exploration of dynamic membrane protein behavior. Molecular dynamics methods enable users to produce stepwise trajectories of proteins on arbitrary spatiotemporal scales that enable the easy identification of dynamic interactions that are beyond the scope of conventional analytical techniques. This article explains the molecular dynamics theoretical framework and popular step-by-step approaches for simulating membrane proteins in planar, and to a lesser extent, nonplanar lipid geometries. We detail popular procedures and computational tools that produce well-packed configurations of lipids and proteins and additionally, the efficient molecular dynamics simulation algorithms that reproduce their dynamic interactions.


Assuntos
Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química
10.
Phys Chem Chem Phys ; 24(11): 6476-6491, 2022 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-35254357

RESUMO

Cellular damage is a key issue in the context of cryopreservation. Much of this damage is believed to be caused by extracellular ice formation at temperatures well above the homogeneous freezing point of pure water. Hence the question: what initiates ice nucleation during cryopreservation? In this paper, we assess whether cellular membranes could be responsible for facilitating the ice nucleation process, and what characteristics would make them good or bad ice nucleating agents. By means of molecular dynamics simulations, we investigate a number of phospholipids and lipopolysaccharide bilayers at the interface with supercooled liquid water. While these systems certainly appear to act as ice nucleating agents, it is likely that other impurities might also play a role in initiating extracellular ice nucleation. Furthermore, we elucidate the factors which affect a bilayer's ability to act as an ice nucleating agent; these are complex, with specific reference to both chemical and structural factors. These findings represent a first attempt to pinpoint the origin of extracellular ice nucleation, with important implications for the cryopreservation process.


Assuntos
Criopreservação , Gelo , Bicamadas Lipídicas , Congelamento , Simulação de Dinâmica Molecular , Água/química
11.
Mol Biol Evol ; 37(7): 1986-2001, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32145025

RESUMO

Genetic variation in the enzymes that catalyze posttranslational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signaling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as an ubiquitin donor for E3 ligases that catalyze ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologs to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse embryonic stem cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extraembryonic primitive endoderm, levels of the PDGFRα and FGFR1 receptor tyrosine kinases were reduced and primitive endoderm differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the receptor tyrosine kinases. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.


Assuntos
Endoderma/enzimologia , Evolução Molecular , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Vertebrados/genética , Substituição de Aminoácidos , Animais , Aurora Quinase B/metabolismo , Feminino , Humanos , Camundongos , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Vertebrados/metabolismo
12.
Chembiochem ; 22(9): 1656-1667, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33411956

RESUMO

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib-rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane-embedded peptides. The protocols presented in this paper open new ground by showing how chiro-optical spectroscopies can throw light on the mechanism of action of AMPs.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Peptídeos Catiônicos Antimicrobianos/química , Dicroísmo Circular , Bicamadas Lipídicas/química , Peptaibols/química , Peptaibols/metabolismo , Fosfatidilcolinas/química , Estereoisomerismo
13.
Annu Rev Phys Chem ; 71: 171-188, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32070216

RESUMO

Gram-negative bacteria are protected by a multicompartmental molecular architecture known as the cell envelope that contains two membranes and a thin cell wall. As the cell envelope controls influx and efflux of molecular species, in recent years both experimental and computational studies of such architectures have seen a resurgence due to the implications for antibiotic development. In this article we review recent progress in molecular simulations of bacterial membranes. We show that enormous progress has been made in terms of the lipidic and protein compositions of bacterial systems. The simulations have moved away from the traditional setup of one protein surrounded by a large patch of the same lipid type toward a more bio-logically representative viewpoint. Simulations with multiple cell envelope components are also emerging. We review some of the key method developments that have facilitated recent progress, discuss some current limitations, and offer a perspective on future directions.


Assuntos
Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Membrana Celular/química , Membrana Celular/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular
14.
J Chem Inf Model ; 61(2): 831-839, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33442985

RESUMO

Hydrogen mass repartitioning (HMR) that permits time steps of all-atom molecular dynamics simulation up to 4 fs by increasing the mass of hydrogen atoms has been used in protein and phospholipid bilayers simulations to improve conformational sampling. Molecular simulation input generation via CHARMM-GUI now supports HMR for diverse simulation programs. In addition, considering ambiguous pH at the bacterial outer membrane surface, different protonation states, either -2e or -1e, of phosphate groups in lipopolysaccharides (LPS) are also supported in CHARMM-GUI LPS Modeler. To examine the robustness of HMR and the influence of protonation states of phosphate groups on LPS bilayer properties, eight different LPS-type all-atom systems with two phosphate protonation states are modeled and simulated utilizing both OpenMM 2-fs (standard) and 4-fs (HMR) schemes. Consistency in the conformational space sampled by standard and HMR simulations shows the reliability of HMR even in LPS, one of the most complex biomolecules. For systems with different protonation states, similar conformations are sampled with a PO41- or PO42- group, but different phosphate protonation states make slight impacts on lipid packing and conformational properties of LPS acyl chains. Systems with PO41- have a slightly smaller area per lipid and thus slightly more ordered lipid A acyl chains compared to those with PO42-, due to more electrostatic repulsion between PO42- even with neutralizing Ca2+ ions. HMR and different protonation states of phosphates of LPS available in CHARMM-GUI are expected to be useful for further investigations of biological systems of diverse origin.


Assuntos
Hidrogênio , Lipopolissacarídeos , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Fosfatos , Reprodutibilidade dos Testes
15.
Int J High Perform Comput Appl ; 35(5): 432-451, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38603008

RESUMO

We develop a generalizable AI-driven workflow that leverages heterogeneous HPC resources to explore the time-dependent dynamics of molecular systems. We use this workflow to investigate the mechanisms of infectivity of the SARS-CoV-2 spike protein, the main viral infection machinery. Our workflow enables more efficient investigation of spike dynamics in a variety of complex environments, including within a complete SARS-CoV-2 viral envelope simulation, which contains 305 million atoms and shows strong scaling on ORNL Summit using NAMD. We present several novel scientific discoveries, including the elucidation of the spike's full glycan shield, the role of spike glycans in modulating the infectivity of the virus, and the characterization of the flexible interactions between the spike and the human ACE2 receptor. We also demonstrate how AI can accelerate conformational sampling across different systems and pave the way for the future application of such methods to additional studies in SARS-CoV-2 and other molecular systems.

16.
Acc Chem Res ; 52(1): 180-188, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30562009

RESUMO

Bacterial membranes, and those of Gram-negative bacteria in particular, are some of the most biochemically diverse membranes known. They incorporate a wide range of lipid types and proteins of varying sizes, architectures, and functions. While simpler biological membranes have been the focus of myriad simulation studies over the years that have yielded invaluable details to complement, and often to direct, ongoing experimental studies, simulations of complex bacterial membranes have been slower to emerge. However, the past few years have seen tremendous activity in this area, leading to advances such as the development of atomistic and coarse-grain models of the lipopolysaccharide (LPS) component of the outer membrane that are compatible with widely used simulation codes. In this Account, we review our contributions to the field of molecular simulations of the bacterial cell envelope, including the development of models of both membranes and the cell wall of Gram-negative bacteria, with a predominant focus on E. coli. At the atomistic level, simulations of chemically accurate models of both membranes have revealed the tightly cross-linked nature of the LPS headgroups and have shown that penetration of solutes through these regions is not as straightforward as the route through phospholipids. The energetic differences between the two routes have been calculated. Simulations of native outer membrane proteins in LPS-containing membranes have shown that the conformational dynamics of the proteins is not only slower in LPS but also different compared to in simpler models of phospholipid bilayers. These chemically more complex and consequently biologically more relevant models are leading to details of conformational dynamics that were previously inaccessible from simulations. Coarse-grain models have enabled simulations of multiprotein systems on time scales of microseconds, leading to insights not only into the rates of protein and lipid diffusion but also into the trends in their respective directions of flow. We find that the motions of LPS molecules are highly correlated with each other but also with outer membrane proteins embedded within the membrane. We have shown that the two leaflets of the outer membrane exhibit communication, whereby regions of low disorder in one leaflet correspond to regions of high disorder in the other. The cell wall remains a comparatively neglected component, although models of the E. coli peptidoglycan are now emerging, particularly at the atomistic level. Our simulations of Braun's lipoprotein have shown that bending and tilting of this protein afford a degree of variability in the gap between the cell wall and the OM. The noncovalent interactions with the cell wall of proteins such as OmpA can further influence the width of this gap by extension or contraction of their linker domains. Overall we have shown that the dynamics of proteins, lipids, and other molecular species within the outer membrane cannot be approximated using simpler phospholipid bilayers, if one is addressing questions regarding the in vivo behavior of Gram-negative bacteria. These membranes have their own unique chemical characteristics that cannot be decoupled from their biological functions.


Assuntos
Membrana Celular/química , Parede Celular/química , Bactérias Gram-Negativas/química , Lipopolissacarídeos/química , Simulação de Dinâmica Molecular , Difusão
17.
J Chem Phys ; 153(4): 044122, 2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32752683

RESUMO

The outer membrane of Gram-negative bacteria is almost exclusively composed of lipopolysaccharide in its outer leaflet, whereas the inner leaflet contains a mixture of phospholipids. Lipopolysaccharide diffuses at least an order of magnitude slower than phospholipids, which can cause issues for molecular dynamics simulations in terms of adequate sampling. Here, we test a number of simulation protocols for their ability to achieve convergence with reasonable computational effort using the MARTINI coarse-grained force-field. This is tested in the context both of potential of mean force (PMF) calculations for lipid extraction from membranes and of lateral mixing within the membrane phase. We find that decoupling the cations that cross-link the lipopolysaccharide headgroups from the extracted lipid during PMF calculations is the best approach to achieve convergence comparable to that for phospholipid extraction. We also show that lateral lipopolysaccharide mixing/sorting is very slow and not readily addressable even with Hamiltonian replica exchange. We discuss why more sorting may be unrealistic for the short (microseconds) timescales we simulate and provide an outlook for future studies of lipopolysaccharide-containing membranes.


Assuntos
Membrana Externa Bacteriana/química , Lipídeos/isolamento & purificação , Bactérias Gram-Negativas/química , Lipídeos/química , Lipopolissacarídeos/química , Simulação de Dinâmica Molecular
18.
Biophys J ; 115(8): 1445-1456, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30287112

RESUMO

Covalent modification of outer membrane lipids of Gram-negative bacteria can impact the ability of the bacterium to develop resistance to antibiotics as well as modulating the immune response of the host. The enzyme LpxR from Salmonella typhimurium is known to deacylate lipopolysaccharide molecules of the outer membrane; however, the mechanism of action is unknown. Here, we employ molecular dynamics and Monte Carlo simulations to study the conformational dynamics and substrate binding of LpxR in representative outer membrane models as well as detergent micelles. We examine the roles of conserved residues and provide an understanding of how LpxR binds its substrate. Our simulations predict that the catalytic H122 must be Nε-protonated for a single water molecule to occupy the space between it and the scissile bond, with a free binding energy of -8.5 kcal mol-1. Furthermore, simulations of the protein within a micelle enable us to predict the structure of the putative "closed" protein. Our results highlight the need for including dynamics, a representative environment, and the consideration of multiple tautomeric and rotameric states of key residues in mechanistic studies; static structures alone do not tell the full story.


Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Cátions/metabolismo , Membrana Celular/enzimologia , Conformação Proteica , Salmonella typhimurium/enzimologia , Acilação , Hidrolases de Éster Carboxílico/genética , Domínio Catalítico , Cátions/química , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica
19.
Biochemistry ; 57(29): 4374-4381, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29949342

RESUMO

Protein bacteriocins are potent narrow spectrum antibiotics that exploit outer membrane porins to kill bacteria by poorly understood mechanisms. Here, we determine how colicins, bacteriocins specific for Escherichia coli, engage the trimeric porin OmpF to initiate toxin entry. The N-terminal ∼80 residues of the nuclease colicin ColE9 are intrinsically unstructured and house two OmpF binding sites (OBS1 and OBS2) that reside within the pores of OmpF and which flank an epitope that binds periplasmic TolB. Using a combination of molecular dynamics simulations, chemical trimerization, isothermal titration calorimetry, fluorescence microscopy, and single channel recording planar lipid bilayer measurements, we show that this arrangement is achieved by OBS2 binding from the extracellular face of OmpF, while the interaction of OBS1 occurs from the periplasmic face of OmpF. Our study shows how the narrow pores of oligomeric porins are exploited by colicin disordered regions for direction-specific binding, which ensures the constrained presentation of an activating signal within the bacterial periplasm.


Assuntos
Colicinas/metabolismo , Escherichia coli/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Porinas/metabolismo , Sítios de Ligação , Colicinas/química , Escherichia coli/química , Escherichia coli/citologia , Proteínas Intrinsicamente Desordenadas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Porinas/química , Ligação Proteica
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