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DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA–Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA–Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4–5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1–2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy ( = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.Significance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter that exports a huge range of compounds out of cells and is thus one of the key proteins in conferring multi-drug resistance in cancer. Understanding how it achieves such a broad specificity and the series of conformational changes that allow export to occur form major, on-going, research objectives around the world. Much of our knowledge to date has been derived from mutagenesis and assay data. However, in recent years, there has also been great progress in structural biology and although the structure of human P-gp has not yet been solved, there are now a handful of related structures on which homology models can be built to aid in the interpretation of the vast amount of experimental data that currently exists. Many models for P-gp have been built with this aim, but the situation is complicated by the apparent flexibility of the system and by the fact that although many potential templates exist, there is large variation in the conformational state in which they have been crystallized. In this review, we summarize how homology modelling has been used in the past, how models are typically selected and finally illustrate how MD simulations can be used as a means to give more confidence about models that have been generated via this approach.

P-glycoprotein (P-gp) can transport a wide range of very different hydrophobic organic molecules across the membrane. Its ability to extrude molecules from the cell creates delivery problems for drugs that target proteins in the central nervous system (CNS) and also causes drug-resistance in many forms of cancer. Whether a drug will be susceptible to export by P-gp is difficult to predict and currently this is usually assessed with empirical and/or animal models. Thus, there is a need to better understand how P-gp works at the molecular level in order to fulfil the 3Rs: Refinement, reduction and replacement of animals in research. As structural information increasingly becomes available, our understanding at the molecular level improves. Proteins like P-gp are however very dynamic entities and thus one of the most appropriate ways to study them is with molecular dynamics simulations, especially as this can capture the influence of the surrounding environment. Recent parameterization developments have meant that it is now possible to simulate lipid bilayers that more closely resemble in vivo membranes in terms of their composition. In this report we construct a complex lipid bilayer that mimics the composition of brain epithelial cells and examine the interactions of it with P-gp. We find that the negatively charged phosphatidylserine lipids in the inner leaflet of the membrane tend to form an annulus around P-gp. We also observed the interaction of cholesterol with three distinct areas of the P-gp. Potential of mean force (PMF) calculations suggest that a crevice between transmembrane helices 10 and 12 has particularly favourable interaction energy for cholesterol.

Permeation of drugs across lipid bilayers is a key factor in dictating how effective they will be. In vivo, the issue is compounded by the presence of drug-exporter proteins such as P-glycoprotein. However, despite intense effort, exactly what controls permeation and susceptibility to export is still poorly understood. In this work we examine two well-studied drugs for which interaction with P-glycoprotein has been studied before: amitriptyline, a known substrate and clozapine, which is not a substrate. Extensive MD simulations, including potential of mean force (PMF) profiles of the compounds in all possible protonation states, reveal that the preferred location of the compounds in different bilayers in different protonation states is remarkably similar. For both molecules in charged states, there is a substantial barrier to crossing the bilayer. Clozapine however, shows an energetic barrier to movement across the bilayer even in a protonation state that results in an uncharged molecule. For amitriptyline there is only a very small barrier of approximately 1.3 kcal mol(-1). Further analysis revealed that the conformational and orientational behavior of the two compounds was also similar, with the sidechain interacting with the lipid headgroups. This effect was much stronger if the sidechain was charged (protonated). These interactions with lipid bilayers were confirmed by NMR ROESY experiments. The results are discussed in terms of their potential interactions with export proteins like P-glycoprotein.

P-glycoprotein (P-gp) can transport a wide range of very different hydrophobic organic molecules across the membrane. Its ability to extrude molecules from the cell creates delivery problems for drugs that target proteins in the central nervous system (CNS) and also causes drug-resistance in many forms of cancer. Whether a drug will be susceptible to export by P-gp is difficult to predict and currently this is usually assessed with empirical and/or animal models. Thus, there is a need to better understand how P-gp works at the molecular level in order to fulfil the 3Rs: Refinement, reduction and replacement of animals in research. As structural information increasingly becomes available, our understanding at the molecular level improves. Proteins like P-gp are however very dynamic entities and thus one of the most appropriate ways to study them is with molecular dynamics simulations, especially as this can capture the influence of the surrounding environment. Recent parameterization developments have meant that it is now possible to simulate lipid bilayers that more closely resemble in vivo membranes in terms of their composition. In this report we construct a complex lipid bilayer that mimics the composition of brain epithelial cells and examine the interactions of it with P-gp. We find that the negatively charged phosphatidylserine lipids in the inner leaflet of the membrane tend to form an annulus around P-gp. We also observed the interaction of cholesterol with three distinct areas of the P-gp. Potential of mean force (PMF) calculations suggest that a crevice between transmembrane helices 10 and 12 has particularly favourable interaction energy for cholesterol.

Due to the wide variety of substrates it extrudes, P-glycoprotein is one of the main causes of multidrug resistance. Although the chemical structure of a ligand is likely to dictate interactions within the binding site, it is equally important to understand how access to the binding site itself is controlled. Access to the binding site is via an aqueous cavity formed in the main by transmembrane helices (TMHs). These TMHs also have helical structure that extends into the cytoplasm, but the hydrophobic vacuum cleaner model suggests that access to the aqueous cavity is via gates between the membranous regions of TMHs 4 and 6 or between TMHs 10 and 12.Previously, we observed that the intra-membrane sections of these gates tend to close while cytoplasmic regions remain open. We have explored this further via the use of steered MD with substrates initially positioned in the cytoplasmic leaflet of the bilayer. Rather unexpectedly, the simulations show that entrance through the intra-membrane gate requires a significantly larger amount of work compared to the cytoplasmic entrance and with a preference for the cavity between TMH 4 and 6 compared to that between TMH 10 and 12.Once the compound has reached the aqueous cavity, it can freely diffuse towards its binding site. The binding mode was independently assessed through the use of consensus docking and appears to be similar to the so-called “R-site”. The docked conformations were then used to calculate possible unbinding pathways of amitriptyline with the Protein Energy Landscape Exploration (PELE) method.Taken together, these combined approaches suggest that a likely access route to the binding site for many compounds is through a cytoplasmic gate formed by TMHs 4 and 6.