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Allosteric Role of Substrate Occupancy Toward the Alignment of P-glycoprotein Nucleotide Binding Domains.

ABSTRACT: P-glycoprotein (Pgp) is an ATP-binding cassette transporter that eliminates toxins from the cell but causes multidrug resistance in chemotherapies. The crystal structures of Pgp revealed drug-like compounds bound to an inward-facing conformation in which the energy-harnessing nucleotide binding domains (NBDs) were widely separated with no interfacial interaction. Following drug binding, inward-facing Pgp must transition to an NBD dimer conformation to achieve ATP binding and hydrolysis at canonical sites defined by both halves of the interface. However, given the high degree of flexibility shown for this transporter, it is difficult to envision how NBDs overcome entropic considerations for achieving proper alignment in order to form the canonical ATP binding site. We explored the hypothesis that substrate occupancy of the polyspecific drug-binding cavity plays a role in the proper alignment of NBDs using computational approaches. We conducted twelve atomistic molecular dynamics (MD) simulations (100-300?ns) on inward-facing Pgp in a lipid bilayer with and without small molecule substrates to ascertain effects of drug occupancy on NBD dimerization. Both apo- and drug-occupied simulations showed NBDs approaching each other compared to the crystal structures. Apo-Pgp reached a pseudo-dimerization in which NBD signature motifs for ATP binding exhibited a significant misalignment during closure. In contrast, occupancy of three established substrates positioned by molecular docking achieved NBD alignment that was much more compatible with a canonical NBD dimerization trajectory. Additionally, aromatic amino acids, known to confer the polyspecific drug-binding characteristic of the internal pocket, may also govern polyspecific drug access to the cavity. The enrichment of aromatics comprising the TM4-TM6 portal suggested a preferential pathway over the aromatic-poor TM10-TM12 for lateral drug entry from the lipid bilayer. Our study also suggested that drug polyspecificity is enhanced due to a synergism between multiple drug-domain interactions involving 36 residues identified in TM1, 5, 6, 7, 11 and 12.

SUBMITTER: Pan L

PROVIDER: S-EPMC6168518 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Allosteric Role of Substrate Occupancy Toward the Alignment of P-glycoprotein Nucleotide Binding Domains.

Pan Lurong L Aller Stephen G SG

Scientific reports 20181002 1

P-glycoprotein (Pgp) is an ATP-binding cassette transporter that eliminates toxins from the cell but causes multidrug resistance in chemotherapies. The crystal structures of Pgp revealed drug-like compounds bound to an inward-facing conformation in which the energy-harnessing nucleotide binding domains (NBDs) were widely separated with no interfacial interaction. Following drug binding, inward-facing Pgp must transition to an NBD dimer conformation to achieve ATP binding and hydrolysis at canoni ...[more]

PMID: 30279588

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Project description:Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of ‘multidimensional mutagenesis’: mutational effects are quantified for multiple molecular phenotypes—here binding and protein abundance—and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering ‘edgetic’ variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.

Dataset Information

Allosteric Role of Substrate Occupancy Toward the Alignment of P-glycoprotein Nucleotide Binding Domains.

Publications

Allosteric Role of Substrate Occupancy Toward the Alignment of P-glycoprotein Nucleotide Binding Domains.

Similar Datasets

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