Conformation Of Loop Research Articles

Can We Ever Develop an Ideal RNA Force Field? Lessons Learned from Simulations of the UUCG RNA Tetraloop and Other Systems.

Molecular dynamics (MD) simulations are an important and well-established tool for investigating RNA structural dynamics, but their accuracy relies heavily on the quality of the employed force field (ff). In this work, we present a comprehensive evaluation of widely used pair-additive and polarizable RNA ffs using the challenging UUCG tetraloop (TL) benchmark system. Extensive standard MD simulations, initiated from the NMR structure of the 14-mer UUCG TL, revealed that most ffs did not maintain the native state, instead favoring alternative loop conformations. Notably, three very recent variants of pair-additive ffs, OL3CP-gHBfix21, DES-Amber, and OL3R2.7, successfully preserved the native structure over a 10 × 20 μs time scale. To further assess these ffs, we performed enhanced sampling folding simulations of the shorter 8-mer UUCG TL, starting from the single-stranded conformation. Estimated folding free energies (ΔG°fold) varied significantly among these three ffs, with values of 0.0 ± 0.6, 2.4 ± 0.8, and 7.4 ± 0.2 kcal/mol for OL3CP-gHBfix21, DES-Amber, and OL3R2.7, respectively. The ΔG°fold value predicted by the OL3CP-gHBfix21 ff was closest to experimental estimates, ranging from -1.6 to -0.7 kcal/mol. In contrast, the higher ΔG°fold values obtained using DES-Amber and OL3R2.7 were unexpected, suggesting that key interactions are inaccurately described in the folded, unfolded, or misfolded ensembles. These discrepancies led us to further test DES-Amber and OL3R2.7 ffs on additional RNA and DNA systems, where further performance issues were observed. Our results emphasize the complexity of accurately modeling RNA dynamics and suggest that creating an RNA ff capable of reliably performing across a wide range of RNA systems remains extremely challenging. In conclusion, our study provides valuable insights into the capabilities of current RNA ffs and highlights key areas for future ff development.

Allosteric inhibition of PTP1B by bromocatechol-chalcone derivatives.

Development of allosteric inhibitors may be a viable strategy to discover hypoglycemic drugs targeting PTP1B. Allosteric inhibitors occupying the BB site that is a hydrophobic pocket restrict the WPD loop in an open conformation, preventing the physiological dephosphorylation reaction. Toward the BB site, sixty bromocatechol-chalcone derivatives were designed and synthesized as allosteric inhibitors of PTP1B against diabetes mellitus. The most potent compound LXQ-87 (C8) inhibited PTP1B noncompetitively with an IC50 value of 1.061±0.202μM. Oral administration of LXQ-87 reduces the fasting blood glucose level and improves glucose tolerance and dyslipidemia in BKS db/db mice suffering from T2DM. LXQ-87 alleviates insulin resistance and promotes cellular glucose uptake by directly binding to intracellular PTP1B.

The conformation of the nSrc specificity-determining loop in the Src SH3 domain is modulated by a WX conserved sequence motif found in SH3 domains.

Cellular signaling networks are modulated by multiple protein-protein interaction domains that coordinate extracellular inputs and processes to regulate cellular processes. Several of these domains recognize short linear motifs, or SLiMs, which are often highly conserved and are closely regulated. One such domain, the Src homology 3 (SH3) domain, typically recognizes proline-rich SLiMs and is one of the most abundant SLiM-binding domains in the human proteome. These domains are often described as quite versatile, and indeed, SH3 domains can bind ligands in opposite orientations dependent on target sequence. Furthermore, recent work has identified diverse modes of binding for SH3 domains and a wide variety of sequence motifs that are recognized by various domains. Specificity is often attributed to the RT and nSrc loops near the peptide-binding cleft in this domain family, particularly for Class I binding, which is defined as RT and nSrc loop interactions with the N-terminus of the ligand. Here, we used the Src and Abl SH3 domains as a model to further investigate the role of the RT and nSrc loops in SH3 specificity. We created chimeric domains with both the RT and nSrc loop sequences swapped between these SH3 domains, and used fluorescence anisotropy assays to test how relative binding affinities were affected for Src SH3- and Abl SH3-specific ligands. We also used Alphafold-Multimer to model our SH3:peptide complexes in combination with molecular dynamics simulations. We identified a position that contributes to the nSrc loop conformation in Src SH3, the amino acid immediately following a highly conserved Trp that creates a hydrophobic pocket critical for SH3 ligand recognition. We defined this as the WX motif, where X = Trp for Src and Cys for Abl. A broad importance of this position for modulating nSrc loop conformation in SH3 domains is suggested by analyses of previously deposited SH3 structures, multiple sequence alignment of SH3 domains in the human proteome, and our biochemical and computational data of mutant Src and Abl SH3 domains. Overall, our work uses experimental approaches and structural modeling to better understand specificity determinants in SH3 domains.

Structural insights into the engagement of lysophosphatidic acid receptor 1 with different G proteins.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophospholipids derived from cell membranes that activate the endothelial differentiation gene family of G protein-coupled receptors. Activation of these receptors triggers multiple downstream signaling cascades through G proteins such as Gi/o, Gq/11, and G12/13. Therefore, LPA and S1P mediate several physiological processes, including cytoskeletal dynamics, neurite retraction, cell migration, cell proliferation, and intracellular ion fluxes. The basis for the G-protein coupling selectivity of EGD receptors, however, remains unknown. Here, we present cryo-electron microscopy structures of LPA-activated LPA1 in complexes with Gi, Gq, and G13 heterotrimers. Comparison of the three LPA1-G protein structures shows clearly different conformations of intracellular loop 2 (ICL2) and ICL3 that are likely induced by the different Gα protein interfaces. Interestingly, this G-protein interface interaction is a common feature of LPA and S1P receptors. Our findings provide clues to understanding the promiscuity of G-protein coupling in the endothelial differentiation gene family.

High-resolution crystal structure of PD-1 in complex with retifanlimab, the FDA-approved immune checkpoint blocking antibody for treating Merkel cell carcinoma

Retifanlimab is a humanized monoclonal antibody that specifically targets programmed cell death protein 1 (PD-1), an essential immune checkpoint that modulates T-cell immune responses. Several anti-PD-1 antibodies have been market-approved, marking a significant advancement in the treatment of diverse tumor types by restoring the T-cell immune response. Recently, the US FDA approved retifanlimab for treating metastatic or recurrent locally advanced Merkel cell carcinoma. We present the crystal structure of PD-1 in complex with the retifanlimab Fab at a resolution of 1.54 Å to elucidate the structural basis for the mechanism of action of this antibody. This work clarifies the detailed interactions and conformational alterations that occur upon antibody binding. The epitope of retifanlimab partially overlaps with the ligand binding site, and its binding induced unique conformations of the flexible loops within PD-1, including BC, C'D, and FG loops, thereby optimizing interactions with the antibody. A thorough analysis of its interaction with PD-1 and other FDA-approved anti-PD-1 antibodies may provide valuable insights into the rational design of enhanced therapies to regulate immune responses in cancer treatment.

Structural modeling of phosphatidylinositol 3-kinase-γ with novel derivatives of stilbenoids

Phosphatidylinositol 3-kinases (PI3K) form a family of lipid kinases that catalyze the phosphorylation of 3-hydroxyl group of the inositol ring of phosphatidylinositol and its derivatives. It is implicated in inflammatory disorders and cancer thus making it an attractive drug target. Crystal structure of human PI3Kγ was taken and structure was completed using MODELLER and validated using PROCHECK. Stilbenoid molecules, piceatannol and resveratrol, were docked to kinase domain of PI3Kγ using AutoDock Vina and docked complexes were subjected to molecular dynamic simulations using Desmond suite of programmes. Based on the structural analysis of these complexes, modified derivatives of the native molecules were designed, docked and molecular dynamic simulations were performed. Kinase domain has a bi-lobar structure with ATP binding site lying in the cleft connecting the two lobes that are primarily composed of 12 α-helices and 8 β-strands. Piceatannol and resveratrol bind at the ATP binding site, with one its rings in a position primarily occupied by adenine of ATP making a hydrogen bond with backbone of Val882. Molecules also make interactions with Lys833 and several isoleucine residues. Interactions with Ser806 appear to be crucial for the loop conformation and compactness. Derivative molecules of stilbenoids also occupy the ATP binding cleft and the chemical modifications result in hydrogen bonded interactions to Glu880, and ionic interactions to Lys833 and Lys808 thereby enhancing their potencies in comparison to native molecules. Biophysical parameters and quality of interactions of stilbenoid derivatives augurs well for development of potent and specific inhibitory molecules against PI3Kγ enzyme.

Leishmania donovani adenylosuccinate synthetase requires IMP for dimerization and organization of the active site.

Adenylosuccinate synthetase (AdSS), which catalyses the GTP-dependent conversion of inosine monophosphate (IMP) and aspartic acid to succinyl-AMP, plays a major role in purine biosynthesis. In some bacterial AdSS, it is implicated that IMP binding is important to organize the active site, but in certain plant AdSS, GTP performs this role. Here, we report that in Leishmania donovani AdSS, IMP binding favoured dimerization, induced greater conformational change and improved the protein stability more than GTP binding. IMP binding, which resulted in a network of hydrogen bonds, stabilized the conformation of active site loops and brought the switch loop to a closed conformation, which then facilitated GTP binding. Our results provide a basis for designing better inhibitors of leishmanial AdSS.

Two conserved arginine residues facilitate C-S bond cleavage and persulfide transfer in Suf family cysteine desulfurases.

Under conditions of oxidative stress or iron starvation, iron-sulfur cluster biogenesis in E. coli is initiated by the cysteine desulfurase, SufS, via the SUF pathway. SufS is a type II cysteine desulfurase that catalyzes the PLP-dependent breakage of an L-cysteine C-S bond to generate L-alanine and a covalent active site persulfide as products. The persulfide is transferred from SufS to SufE and then to the SufBC2D complex, which utilizes it in iron-sulfur cluster biogenesis. Several lines of evidence suggest two conserved arginine residues that line the solvent side of the SufS active site could be important for function. To investigate the mechanistic roles of R56 and R359, the residues were substituted using site-directed mutagenesis to obtain R56A/K and R359A/K SufS variants. Steady state kinetics indicated R56 and R359 have moderate defects in the desulfurase half reaction but major defects in the transpersulfurase step. Fluorescence polarization binding assays showed that the loss of activity was not due to a defect in forming the SufS/SufE complex. Structural characterization of R56A SufS shows loss of electron density for the α3-α4 loop at the R56/G57 positions, consistent with a requirement of R56 for proper loop conformation. The structure of R359A SufS exhibits a conformational change in the α3-α4 loop allowing R56 to enter the active site and mimics the residue's position in the PLP-cysteine aldimine structure. Taken together, the kinetic, binding, and structural data support a mechanism where R359 plays a role in linking SufS catalysis with modulation of the α3-α4 loop to promote a close-approach interaction of SufS and SufE conducive to persulfide transfer.

Core enhancers of the 3'RR optimize IgH nuclear position and loop conformation for successful oriented class switch recombination.

In B lymphocytes, class switch recombination (CSR) is an essential process that adapts immunoglobulin (Ig) subtypes to antigenresponse. Taking place within the Ig heavy chain (IgH) locus, CSR needs controlled transcription of targeted regions governed by the IgH 3'regulatory region (3'RR). This super-enhancer is composed of four core enhancers surrounded by inverted repeated sequences, forming a quasi-palindrome. In addition to transcription, nuclear organization appears to be an important level in CSR regulation. While it is now established that chromatin loop extrusion takes place within IgH locus to facilitate CSR by bringing the donor and acceptor switch regions closer together, the underlying mechanism that triggers CSR loop formation remains partially understood. Here, by combining DNA 3Dfluorescence in situhybridizationwith various high-throughput approaches, we deciphered critical functions for the 3'RR core enhancer element in nuclear addressing, accessibility and chromatin looping of the IgH locus. We conclude thatthe 3'RR core enhancers are necessary and sufficient to pre-organize the position and conformation of IgH loci in resting B-cell nuclei to enable the deletional recombination events required for productive successful CSR in activated B-cell nuclei.

Cocrystallization of the Src-Family Kinase Hck with the ATP-Site Inhibitor A-419259 Stabilizes an Extended Activation Loop Conformation.

Hematopoietic cell kinase (Hck) is a member of the Src kinase family and is a promising drug target in myeloid leukemias. Here, we report the crystal structure of human Hck in complex with the pyrrolopyrimidine inhibitor A-419259, determined at a resolution of 1.8 Å. This structure reveals the complete Hck active site in the presence of A-419259, including the αC-helix, the DFG motif, and the activation loop. A-419259 binds at the ATP-site of Hck and induces an overall closed conformation of the kinase with the regulatory SH3 and SH2 domains bound intramolecularly to their respective internal ligands. A-419259 stabilizes the DFG-in/αC-helix-out conformation observed previously with Hck and the pyrazolopyrimidine inhibitor PP1 (PDB: 1QCF). However, the activation loop conformations are distinct, with PP1 inducing a folded loop structure with the tyrosine autophosphorylation site (Tyr416) pointing into the ATP binding site, while A-419259 stabilizes an extended loop conformation with Tyr416 facing out into the solvent. Autophosphorylation also induces activation loop extension and significantly reduces the Hck sensitivity to PP1 but not A-419259. In cancer cells where Hck is constitutively active, the extended autophosphorylation loop may render Hck more sensitive to inhibitors like A-419259 which prefer this kinase conformation. More generally, these results provide additional insight into targeted kinase inhibitor design and how conformational preferences of inhibitors may impact selectivity and potency.

A Conserved Tryptophan (Trp10) at the Hydrophobic Core Modulates the Stability and Inhibitory Activity of Potato I Type Inhibitors.

Different inhibitor families have their own conserved three-dimensional structures, but how these structures determine whether a protein can become an inhibitor is still unknown. The buckwheat trypsin inhibitor (BTI) pertains to the Potato I type inhibitor family, which is a simple and essential bio-molecule that serves as a model for the investigation of protease-inhibitor interaction. To study the effects of mutations at Trp10 and Ile25 in the hydrophobic cavity (scaffold) of rBTI on its inhibitory activity and stability. Site-directed mutagenesis and molecular modeling were performed using the sequence of BTI. The hydrogen bonds formed by all amino acids and conformational differences of Trp53 were analyzed in the tertiary structures of rBTI and mutants. Mutant rBTI-W10A almost completely lost its inhibitory activity (retaining 10%), while rBTI-I25A retained about 50% of its inhibitory activity. Both rBTI-W10A and rBTI-I25A could be degraded by trypsin. The hydrogen bond analysis results showed that mutating Trp10 or Ile25 weakened the specific cohesion interactions in the hydrophobic core of rBTI, disrupting the tight hydrogen bond network in the cavity. This further led to difficulty in maintaining the binding loop conformation, ultimately causing the Trp53 to undergo conformational changes. It was also difficult for residues in the mutants to form hydrogen bonds with amino acids in bovine trypsin; thus, the mutants could not stably bind to trypsin. Our findings suggest that the hydrophobic core is also an important factor in the maintenance of inhibitory activity and stability of rBTI.

Structural insights into the diversity and DNA cleavage mechanism of Fanzor

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)8-Barrel Enzyme of Histidine Biosynthesis HisF.

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the β-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the β3-β4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Cytosine analogues as DNA methyltransferase substrates.

DNA methyltransferases are drug targets for myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML)and possibly β-hemoglobinopathies. We characterize the interaction of nucleoside analogues in DNA with a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model for mammalian DNMT1 methyltransferases. We tested DNA containing 5-hydroxymethylcytosine (5hmC), 5-hydroxycytosine (5OHC), 5-methyl-2-pyrimidinone (in the ribosylated form known as 5-methylzebularine, 5mZ), 5,6-dihydro-5-azacytosine (dhaC), 5-fluorocytosine (5FC), 5-chlorocytosine (5ClC), 5-bromocytosine (5BrC)and 5-iodocytosine (5IC). Covalent complex formation was by far most efficient for 5FC. Non-covalent complexes were most abundant for dhaC and 5mZ. Surprisingly, we observed methylation of 5IC and 5BrC, and to a lesser extent 5ClC and 5FC, in the presence, but not the absence of small molecule thiol nucleophiles. For 5IC and 5BrC, we demonstrated by mass spectrometry that the reactions were due to methyltransferase driven dehalogenation, followed by methylation. Crystal structures of M.MpeI-DNA complexes capture the 'in' conformation of the active site loop for analogues with small or rotatable (5mZ) 5-substituents and its 'out' form for bulky 5-substituents. Since very similar 'in' and 'out' loop conformations were also observed for DNMT1, it is likely that our conclusions generalize to other DNA methyltransferases.

The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA’s association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

Roles of the gate loop in β-arrestin-1 conformational dynamics and phosphorylated receptor interaction

Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in arrestins is a critical region for both stabilization of the basal state and interaction with phosphorylated receptors. We investigated the roles of specific residues in the gate loop (K292, K294, and H295) using β-arrestin-1 and phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) as a model system. We measured the binding affinity of V2Rpp and analyzed conformational dynamics of β-arrestin-1. Our results suggest that K294 plays a critical role in the interaction with V2Rpp without influencing the overall conformation of the V2Rpp-bound state. The residues K292 and H295 contribute to the stability of the polar core in the basal state and form a specific conformation of the finger loop in the V2Rpp-bound state.

Loops are geometric catalysts for DNA integration.

The insertion of DNA elements within genomes underpins both genetic diversity and disease when unregulated. Most of DNA insertions are not random and the physical mechanisms underlying the integration site selection are poorly understood. Here, we perform Molecular Dynamics simulations to study the insertion of DNA elements, such as viral DNA or transposons, into naked DNA or chromatin substrates. More specifically, we explore the role of loops within the polymeric substrate and discover that they act as 'geometric catalysts' for DNA integration by reducing the energy barrier for substrate deformation. Additionally, we discover that the 1D pattern and 3D conformation of loops have a marked effect on the distribution of integration sites. Finally, we show that loops may compete with nucleosomes to attract DNA integrations. These results may be tested in vitro and they may help to understand patterns of DNA insertions with implications in genome evolution and engineering.

An alternative conformation of the N-terminal loop of human dihydroorotate dehydrogenase drives binding to a potent antiproliferative agent.

Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine-biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host-targeted antiviral therapy. A molecular exploration of its inhibitor-binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well-established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X-ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine-based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor-binding site is modulated by the presence of three water molecules, thus fine-tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen-bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer Kd/logD7.4 balance and identify membrane-permeable molecules with a drug-like profile in terms of water solubility.