Molecular dynamics analysis of the engrailed homeodomain-DNA recognition

Molecular dynamics (MD) simulations were performed for investigating the role of Gln50 in the engrailed homeodomain-DNA recognition. Employing the crystal structure of free engrailed homeodomain and homeodomain-DNA complex as a starting structure, we carried out MD simulations of: (i) the complex between engrailed homeodomain and a 20 base-pair DNA containing TAATTA core sequence; (ii) the free engrailed homeodomain. The simulations show that homeodomain flexibility does not depend on its ligation state. The engrailed homeodomain shows similar flexibility, and the recognition helix-3 shows very similar characteristic of high rigidity and limited conformational space in two complexation states. At the same time, DNA structure has also no obvious conformational fluctuations. These results preclude the possibility of the side chain of Gln50 forming direct hydrogen bonds to the core DNA bases. MD simulations confirm a few well-conserved sites for water-mediated hydrogen bonds from protein to DNA are occupied by water molecules, and Gln50 interacts with corresponding core DNA bases through water-mediated hydrogen bonds. So Gln50 plays a relatively modest role in determining the affinity and specificity of the engrailed homeodomain. In addition, the electrostatic interaction between homeodomain and phosphate backbone of the DNA is a main factor for N- and C-terminal arm becoming ordered upon DNA binding.     

Molecular dynamics simulations and free energy calculations of netropsin and distamycin binding to an AAAAA DNA binding site

Molecular dynamics simulations have been performed on netropsin in two different charge states and on distamycin binding to the minor groove of the DNA duplex d(CGCGAAAAACGCG)·d(CGCGTTTTTCGCG). The relative free energy of binding of the two non-covalently interacting ligands was calculated using the thermodynamic integration method and reflects the experimental result. From 2 ns simulations of the ligands free in solution and when bound to DNA, the mobility and the hydrogen-bonding patterns of the ligands were studied, as well as their hydration. It is shown that even though distamycin is less hydrated than netropsin, the loss of ligand–solvent interactions is very similar for both ligands. The relative mobilities of the ligands in their bound and free forms indicate a larger entropic penalty for distamycin when binding to the minor groove compared with netropsin, partially explaining the lower binding affinity of the distamycin molecule. The detailed structural and energetic insights obtained from the molecular dynamics simulations allow for a better understanding of the factors determining ligand–DNA binding.     

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA-binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN[downward arrow]NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN[downward arrow]NGGC), and both enzymes cleave their target DNAs to leave 3-base 3' overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.     

The binding process of a nonspecific enzyme with DNA

Protein-DNA recognition of a nonspecific complex is modeled to understand the nature of the transient encounter states. We consider the structural and energetic features and the role of water in the DNA grooves in the process of protein-DNA recognition. Here we have used the nuclease domain of colicin E7 (N-ColE7) from Escherichia coli in complex with a 12-bp DNA duplex as the model system to consider how a protein approaches, encounters, and associates with DNA. Multiscale simulation studies using Brownian dynamics and molecular-dynamics simulations were performed to provide the binding process on multiple length- and timescales. We define the encounter states and identified the spatial and orientational aspects. For the molecular length-scales, we used molecular-dynamics simulations. Several intermediate binding states were found, which have different positions and orientations of protein around DNA including major and minor groove orientations. The results show that the contact number and the hydrated interfacial area are measures that facilitate better understanding of sequence-independent protein-DNA binding landscapes and pathways.     

Statistical analysis of structural determinants for protein–DNA‐binding specificity

DNA‐binding proteins play critical roles in biological processes including gene expression, DNA packaging and DNA repair. They bind to DNA target sequences with different degrees of binding specificity, ranging from highly specific (HS) to nonspecific (NS). Alterations of DNA‐binding specificity, due to either genetic variation or somatic mutations, can lead to various diseases. In this study, a comparative analysis of protein–DNA complex structures was carried out to investigate the structural features that contribute to binding specificity. Protein–DNA complexes were grouped into three general classes based on degrees of binding specificity: HS, multispecific (MS), and NS. Our results show a clear trend of structural features among the three classes, including amino acid binding propensities, simple and complex hydrogen bonds, major/minor groove and base contacts, and DNA shape. We found that aspartate is enriched in HS DNA binding proteins and predominately binds to a cytosine through a single hydrogen bond or two consecutive cytosines through bidentate hydrogen bonds. Aromatic residues, histidine and tyrosine, are highly enriched in the HS and MS groups and may contribute to specific binding through different mechanisms. To further investigate the role of protein flexibility in specific protein–DNA recognition, we analyzed the conformational changes between the bound and unbound states of DNA‐binding proteins and structural variations. The results indicate that HS and MS DNA‐binding domains have larger conformational changes upon DNA‐binding and larger degree of flexibility in both bound and unbound states. Proteins 2016; 84:1147–1161. © 2016 Wiley Periodicals, Inc.     

Role of base flipping in specific recognition of damaged DNA by repair enzymes

DNA repair enzymes induce base flipping in the process of damage recognition. Endonuclease V initiates the repair of cis, syn thymine dimers (TD) produced in DNA by UV radiation. The enzyme is known to flip the base opposite the damage into a non-specific binding pocket inside the protein. Uracil DNA glycosylase removes a uracil base from G.U mismatches in DNA by initially flipping it into a highly specific pocket in the enzyme. The contribution of base flipping to specific recognition has been studied by molecular dynamics simulations on the closed and open states of undamaged and damaged models of DNA. Analysis of the distributions of bending and opening angles indicates that enhanced base flipping originates in increased flexibility of the damaged DNA and the lowering of the energy difference between the closed and open states. The increased flexibility of the damaged DNA gives rise to a DNA more susceptible to distortions induced by the enzyme, which lowers the barrier for base flipping. The free energy profile of the base-flipping process was constructed using a potential of mean force representation. The barrier for TD-containing DNA is 2.5 kcal mol(-1) lower than that in the undamaged DNA, while the barrier for uracil flipping is 11.6 kcal mol(-1) lower than the barrier for flipping a cytosine base in the undamaged DNA. The final barriers for base flipping are approximately 10 kcal mol(-1), making the rate of base flipping similar to the rate of linear scanning of proteins on DNA. These results suggest that damage recognition based on lowering the barrier for base flipping can provide a general mechanism for other DNA-repair enzymes.     

Role of structure and dynamics of DNA with cisplatin and oxaliplatin adducts in various sequence contexts on binding of HMGB1a

Anti-cancer drugs, such as cisplatin and oxaliplatin, covalently bind to adjacent guanine bases in DNA to form intra-strand adducts. Differential recognition of drug–DNA adducts by the protein HMGB1a has been related to the differences in efficacy of these drugs in tumours. Additionally, the bases flanking the adduct (the sequence context) also have a marked effect on HMGB1a binding affinity. We perform atomistic molecular dynamics simulations of DNA with cisplatin and oxaliplatin adducts in four sequence contexts (AGGC, CGGA, TGGA and TGGT) in the absence and presence of HMGB1a. The structure of HMGB1a-bound drug–DNA molecules is independent of sequence and drug identity, confirming that differential recognition cannot be explained by the protein-bound structure. The differences in the static and conformational dynamics of the drug–DNA structures in the absence of the protein explain some but not all trends in differential binding affinity of HMGB1a. Since the minor groove width and helical bend of all drug–DNA molecules in the unbound state are lower than the protein-bound state, HMGB1a must actively deform the DNA during binding. The thermodynamic pathway between the unbound and protein-bound states could be an additional factor in the binding affinity of HMGB1a for drug–DNA adducts in various sequence contexts.     

Nucleotide binding switches the information flow in ras GTPases

The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (S(GDP) and S(GTP), respectively). For all the considered systems, the intrinsic flexibility of S(GDP) was higher than that of S(GTP), suggesting that Guanine Exchange Factor (GEF) recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Gα proteins, especially in the inactive state. Indeed, S(GDP) of Gα(t), is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins), compared to GTP.     

Multiple substrate binding states and chiral recognition in cofactor-independent glutamate racemase: a molecular dynamics study

Glutamate racemase (MurI) catalyzes the racemization of glutamate; two cysteine residues serve as catalytic acid and base. On the basis of the crystal structure of MurI from the hyperthermophilic bacterium Aquifex pyrophilus, we performed molecular dynamics (MD) simulations of six different systems to investigate stereochemistry, substrate ligation, and active site protonation state. The catalytic competence of individual systems was assessed by the abundance of reactive conformers. Only systems in which Cys70 is poised to deprotonate d-Glu were found to be catalytically competent (idem Cys178/l-Glu), in agreement with the experimentally observed stereochemistry of Lactobacillus fermentii MurI [Tanner, M. E. et al. (1993) Biochemistry 32, 3998-4006]. Only systems in which the alpha-amino group of l/d-Glu and the imidazole moiety of His are deprotonated are catalytically competent. The active site of MurI displays an unusual flexibility in substrate ligation, and several transitions between stable binding patterns were observed. In catalytically competent binding states, the conserved threonine residues 72, 114, and 117 ligate the alpha-carboxylate of Glu and the Asn71 amides ligate the alpha-amino group of Glu, whereas the delta-carboxylate of Glu is steered by electrostatic repulsion from the Asp7 and Glu147 side chain carboxylates. A network of hydrogen bonds controls the positioning of each thiol/thiolate. In what we term substrate flipping, Glu suddenly rotates into a binding pattern that resembles the post-racemization state of the other enantiomer, i.e., each enantiomer can be bound in two distinct states. Substrate flipping and unfavorable substrate binding successively trigger dissociation of the substrate, accompanied by an opening of the active site channel. We explain how the weak binding of Glu contributes to catalysis and suggest a mechanism by which binding mismatches are propagated into an opening of the active site.     

Subunit exchange and the role of dimer flexibility in DNA binding by the Fis protein

Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA.     

Conformational flexibility of the leucine binding protein examined by protein domain coarse-grained molecular dynamics

Periplasmic binding proteins are the initial receptors for the transport of various substrates over the inner membrane of gram-negative bacteria. The binding proteins are composed of two domains, and the substrate is entrapped between these domains. For several of the binding proteins it has been established that a closed-up conformation exists even without substrate present, suggesting a highly flexible apo-structure which would compete with the ligand-bound protein for the transporter interaction. For the leucine binding protein (LBP), structures of both open and closed conformations are known, but no closed-up structure without substrate has been reported. Here we present molecular dynamics simulations exploring the conformational flexibility of LBP. Coarse grained models based on the MARTINI force field are used to access the microsecond timescale. We show that a standard MARTINI model cannot maintain the structural stability of the protein whereas the ELNEDIN extension to MARTINI enables simulations showing a stable protein structure and nanosecond dynamics comparable to atomistic simulations, but does not allow the simulation of conformational flexibility. A modification to the MARTINI-ELNEDIN setup, referred to as domELNEDIN, is therefore presented. The domELNEDIN setup allows the protein domains to move independently and thus allows for the simulation of conformational changes. Microsecond domELNEDIN simulations starting from either the open or the closed conformations consistently show that also for LBP, the apo-structure is flexible and can exist in a closed form.

Stability and sugar recognition ability of ricin-like carbohydrate binding domains

Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two β-trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained a detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1α and site 2γ recognition.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

DNA-binding interactions and conformational fluctuations of Tc3 transposase DNA binding domain examined with single molecule fluorescence spectroscopy

The fluorescent dye tetramethylrhodamine (TMR) was conjugated to a synthetic peptide containing the sequence-specific DNA binding domain of Tc3 transposase. Steady-state and single molecule fluorescence spectroscopy was used to investigate protein conformational fluctuations and the thermodynamics of binding interactions. Evidence is presented to show that the TMR-Tc3 conjugate exists in at least two conformational states. The most stable conformation is one in which the TMR fluorescence is quenched. Upon binding to DNA, the total fluorescence from TMR-Tc3 increases by three- to fourfold. Single molecule measurements of TMR-Tc3 bound to DNA shows that this complex also fluctuates between a fluorescent and quenched form. The fluorescent form of the conjugate is stabilized when bound to DNA, and this accounts for part of the increase in total fluorescence. In addition, the inherent photodynamics of the dye itself is also altered (e.g., fluorescent lifetime or triplet yield) in such a way that the total fluorescence from the conjugate bound to DNA is enhanced relative to the unbound form.