L1 Loop Research Articles

Exploring the Genetic and Functional Diversity of Porphyromonas gingivalis Survival Factor RagAB

Porphyromonas gingivalis is a key pathogen in periodontitis. Its outer membrane contains the RagAB transport complex, which has been implicated in protein uptake, essential for a proteolytic species. RagA is a 22-stranded β-barrel, and RagB is the corresponding 4-TPR lid, together forming a TonB-dependent system acting as a “pedal bin”. Four different alleles were observed, of which ragAB-1 is more virulent than the others. Our aim was to map ragAB in 129 strains of P. gingivalis and related species available in our collection, supported by a newly introduced universal PCR for amplification/sequencing of all four ragA variants and to find reasons for the differences in virulence and/or fitness. Regarding the PCR method, by pairing established Long-PCR primers with our newly designed sequencing primers (ragA-F0, -F1, -R2, -R2a, -R4), it was possible to amplify and sequence all four ragA variants. The same was not possible for ragB due to high heterogeneity. The mapping allowed us to type all strains into ragAB-1-4. For each type, some strains (of mainly animal origin such as Porphyromonas gulae) with slightly different amino acid sequences were identified (designated ragAB-1a to -4a). In terms of function, the transfer of recently discovered SusCD information to the similar RagAB complex provided new insights. Substrate specificity as well as length of pedal could be the route to differential virulence (survival rate, fitness) as Rag-1 (closer related to Rag-3/4) and Rag-2 were found to be massively different here. In general, substrate–ligand-binding sites seem to be quite variable with the exception of Rag-1, probably indicating nutritional preferences. In addition, an insertion (8 aa long) found in loop L7 throughout RagA-2 could not only affect the dynamics of lid opening/closing but might also alter the associated substrate throughput rate.

Unraveling antibody-induced structural dynamics in the ADAMTS13 CUB1-2 domains via HDX-MS.

Allosteric regulation of ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type-1 motif, member 13) activity involves an interaction between its Spacer (S) and CUB1-2 domains to keep the enzyme in a closed, latent conformation. Monoclonal antibodies (mAb) uncouple the S-CUB interaction to open the ADAMTS13 conformation and thereby disrupt the global enzyme latency. The molecular mechanism behind this mAb-induced allostery remains poorly understood. To gain insights in the mAb-induced S-CUB uncoupling and global latency disruption, we combined hydrogen/deuterium exchange mass spectrometry (HDX MS) with structural analysis of ADAMTS13 CUB1-2 mutants. Thereby, the CUB1 L3 and L9 loops were fine-mapped as the 17G2 mAb binding epitope. Indirect S-CUB uncoupling was observed as mAb binding induced extensive structural dynamics within both CUB1-2 domains without directly targeting the contiguous CUB1 surface that engages with the ADAMTS13 S domain. HDX MS analysis revealed the short interdomain linker to structurally cover the central CUB1-2 domain interface, which also showed some protein regions that became more exposed upon mAb binding. Therefore, repositioning of the central CUB1-2 interface appears crucial to transfer structural dynamics between both domains. Nevertheless, mutagenesis of the short linker did not disrupt the ADAMTS13 global latency as its closed conformation was preserved. Presumably, allosteric disruption of the global latency requires a structural impact extending beyond the central interface repositioning. As anti-ADAMTS13 autoantibodies from immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients also induce an open ADAMTS13 conformation, our novel insights in the antibody-mediated global latency disruption boost our understanding of the iTTP disease pathology.

Structural comparison of substrate-binding pockets of serine β-lactamases in classes A, C, and D.

β-lactams have been the most successful antibiotics, but the rise of multi-drug resistant (MDR) bacteria threatens their effectiveness. Serine β-lactamases (SBLs), among the most common causes of resistance, are classified as A, C, and D, with numerous variants complicating structural and substrate spectrum comparisons. This study compares representative SBLs of these classes, focusing on the substrate-binding pocket (SBP). SBP is kidney bean-shaped on the indented surface, formed mainly by loops L1, L2, and L3, and an additional loop Lc in class C. β-lactams bind in a conserved orientation, with the β-lactam ring towards L2 and additional rings towards the space between L1 and L3. Structural comparison shows each class has distinct SBP structures, but subclasses share a conserved scaffold. The SBP structure, accommodating complimentary β-lactams, determines the substrate spectrum of SBLs. The systematic comparison of SBLs, including structural compatibility between β-lactams and SBPs, will help understand their substrate spectrum.

The Effect of Chalcogen-Chalcogen Bond Formation in the New Delhi Metallo-β-Lactamase 1 Enzyme to Counteract Antibiotic Resistance.

New Delhi metallo-β-lactamase 1 (NDM-1) is an enzyme involved in the drug resistance of many bacteria against most of the widely adopted antibiotics, such as penicillins, cephalosporins, and carbapenems. Consequently, inhibiting NDM-1 swiftly has gained significant interest as a strategy to counteract this bacterial defense mechanism, thereby restoring the effectiveness of antibiotics. Among the inhibitors tested against the enzyme, ebselen (EbSe) showed particularly promising results. This molecule, renowned for its numerous benefits to the human body, targets the enzyme's active site at Cys208 with its selenium atom, facilitating the expulsion of the catalytic zinc ion from the active pocket. Since the inhibitory mechanism of EbSe remains poorly understood, gaining detailed information about it is highly desirable. In the present work, density functional theory calculations and μs-long molecular dynamics simulations are carried out to investigate the reaction mechanism of EbSe with NDM-1, unveiling the structural implications of the inhibition. A large model of the NDM-1 active site is built to investigate the different mechanistic proposals for the SeEbSe-SCys208 bond formation. Deeper insights into Lys211 are also provided to consolidate its role during the inhibition process. Furthermore, the chemical reaction with the ebsulfur (EbS) molecule is also investigated to compare its behavior with that of the periodic relative selenium. Molecular dynamics simulations, besides evidencing the role of the L3 and L10 loops in the occurrence of the inhibition, corroborate the Zn ion release from the active site as a result of the complete disruption of its coordination sphere caused by the creation of the SeEbSe-SCys208 covalent bond.

Heterozygous and generalist MxA super-restrictors overcome breadth-specificity tradeoffs in antiviral restriction.

Antiviral restriction factors such as MxA (myxovirus resistance protein A) inhibit a broad range of viruses. However, they face the challenge of maintaining this breadth as viruses evolve to escape their defense. Viral escape drives restriction factors to evolve rapidly, selecting for amino acid changes at their virus-binding interfaces to regain defense. How do restriction factors balance the breadth of antiviral functions against the need to evolve specificity against individual escaping viruses? We explored this question in human MxA, which uses its rapidly evolving loop L4 as the specificity determinant for orthomyxoviruses such as THOV and IAV. Previous combinatorial mutagenesis of rapidly evolving residues in human MxA loop L4 revealed variants with a ten-fold increase in potency against THOV. However, this strategy did not yield improved IAV restriction, suggesting a strong tradeoff between antiviral specificity and breadth. Here, using a modified combinatorial mutagenesis strategy, we find 'super-restrictor' MxA variants with over ten-fold enhanced restriction of the avian IAV strain H5N1 but reduced THOV restriction. Analysis of super-restrictor MxA variants reveals that the identity of residue 561 explains most of MxA's breadth-specificity tradeoff in H5N1 versus THOV restriction. However, rare 'generalist' super-restrictors with enhanced restriction of both viruses allow MxA to overcome the breadth-specificity tradeoff. Finally, we show that a heterozygous combination of two 'specialist' super-restrictors, one against THOV and the other against IAV, enhances restriction against both viruses. Thus, two strategies enable restriction factors such as MxA to increase their restriction of diverse viruses to overcome breadth-specificity tradeoffs that may be pervasive in host-virus conflicts.

CDR L3 Loop Rearrangement Switches Multispecific SPE-7 IgE Antibody From Hapten to Protein Binding.

The monoclonal IgE antibody SPE-7 was originally raised against a 2,4-dinitrophenyl (DNP) target. Through its ability to adopt multiple conformations, the antibody is capable of binding to a diverse range of small haptens and large proteins. The present study examines a dataset of experimentally determined crystal structures of the SPE-7 antibody to gain insight into the mechanisms that contribute to its multispecificity. With the emergence of more and more therapeutic antibodies against a huge repertoire of different targets, our research could be of great interest for future drug development. We are able to discriminate between the different paratope-binding states in the conformational ensembles obtained by enhanced sampling molecular dynamics simulations, and to calculate their transition timescales and state probabilities. Furthermore, we describe the key residues responsible for discriminating between the different binding capacities and identify a tryptophan in a central position of the CDR L3 loop as the residue of greatest interest. The overall dynamics of the paratope appear to be mainly influenced by the CDR L3 and CDR L1 loops.

Molecular Mechanism of Ciprofloxacin Translocation Through the Major Diffusion Channels of the ESKAPE Pathogens Klebsiella pneumoniae and Enterobacter cloacae.

Experimental studies on the translocation and accumulation of antibiotics in Gram-negative bacteria have revealed details of the properties that allow efficient permeation through bacterial outer membrane porins. Among the major outer membrane diffusion channels, OmpF has been extensively studied to understand the antibiotic translocation process. In a few cases, this knowledge has also helped to improve the efficacy of existing antibacterial molecules. However, the extension of these strategies to enhance the efficacy of other existing and novel drugs require comprehensive molecular insight into the permeation process and an understanding of how antibiotic and channel properties influence the effective permeation rates. Previous studies have investigated how differences in antibiotic charge distribution can influence the observed permeation pathways through the OmpF channel, and have shown that the dynamics of the L3 loop can play a dominant role in the permeation process. Here, we perform all-atom simulations of the OmpF orthologs, OmpE35 from Enterobacter cloacae and OmpK35 from Klebsiella pneumoniae. Unbiased simulations of the porins and biased simulations of the ciprofloxacin permeation processes through these channels provide insight into the differences in the permeation pathway and energetics. In addition, we show that similar to the OmpF channel, antibiotic-induced dynamics of the L3 loop are also operative in the orthologs. However, the sequence and structural differences, influence the extent of the L3 loop fluctuations with OmpK35 showing greater stability in unbiased runs and subdued fluctuations in simulations with ciprofloxacin.

Conformations of KRAS4B Affected by Its Partner Binding and G12C Mutation: Insights from GaMD Trajectory-Image Transformation-Based Deep Learning.

Binding of partners and mutations highly affects the conformational dynamics of KRAS4B, which is of significance for deeply understanding its function. Gaussian accelerated molecular dynamics (GaMD) simulations followed by deep learning (DL) and principal component analysis (PCA) were carried out to probe the effect of G12C and binding of three partners NF1, RAF1, and SOS1 on the conformation alterations of KRAS4B. DL reveals that G12C and binding of partners result in alterations in the contacts of key structure domains, such as the switch domains SW1 and SW2 together with the loops L4, L5, and P-loop. Binding of NF1, RAF1, and SOS1 constrains the structural fluctuation of SW1, SW2, L4, and L5; on the contrary, G12C leads to the instability of these four structure domains. The analyses of free energy landscapes (FELs) and PCA also show that binding of partners maintains the stability of the conformational states of KRAS4B while G12C induces greater mobility of the switch domains SW1 and SW2, which produces significant impacts on the interactions of GTP with SW1, L4, and L5. Our findings suggest that partner binding and G12C play important roles in the activity and allosteric regulation of KRAS4B, which may theoretically aid in further understanding the function of KRAS4B.

Molecular basis of facilitated target search and sequence discrimination of TALE homeodomain transcription factor Meis1

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD–DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease.

Molecular characteristics of fowl adenovirus strains detected in broiler chickens on diets without immunostimulant supplements.

Outbreaks of fowl adenovirus (FAdV) infection in chicken flocks in Poland threaten birds' health and lives and are rising in frequency. The risk of these infections in immunocompromised poultry flocks with developed clinical symptoms was analysed through virus detection in broiler chicks and correlation of cases with the birds' immune strength. Samples were analysed from four broiler farms with chicks from the same hatchery in Silesia, Poland where feeding regimes were different. A normal diet was provided to birds on the control farm; a normal diet and probiotic, prebiotic, vitamin and microelement supplementation was supplied on another farm; a normal diet and antibiotics on the third; and a normal diet and both forms of supplementation were given on the fourth farm. Amplification of the virus DNA in a PCR with hexon gene L1 loop hypervariable region 1-4 primers determined the molecular characteristics of isolates of adenovirus strains obtained from necropsy tissue samples. The amplicon sequences were analysed, the pair-wise distances were determined, the maximum likelihood estimate for the gamma parameter for site rates was produced, Tajima's D neutrality test was run and the relative synonymous codon usage and transition/transversion bias were calculated. Two species and two serotypes of fowl adenovirus - MW353018-FAdV-1/A-L-liver and MW353019-FAdV-5/B-I-intestine - were isolated in three-week-old broiler chicks on the control farm. Supplementation of broiler chicken flocks with probiotics, prebiotics, vitamins and microelements may have a significant beneficial effect on immunity and can prevent virus infection. The studies provided new information on the molecular characteristics of adenovirus strains isolated from chicks with a low level of immunity.

NMR insights into β-Lactamase activity of UVI31+ Protein from Chlamydomonas reinhardtii

β-Lactamases (EC 3.5.2.6) confer resistance against β-lactam group-containing antibiotics in bacteria and higher eukaryotes, including humans. Pathogenic bacterial resistance against β-lactam antibiotics is a primary concern for potential therapeutic developments and drug targets. Here, we report putative β-lactamase activity, sulbactam binding (a β-lactam analogue) in the low μM affinity range, and site-specific interaction studies of a 14 kDa UV- and dark-inducible protein (abbreviated as UVI31+, a BolA homologue) from Chlamydomonas reinhartii. Intriguingly, the solution NMR structure of UVI31 + bears no resemblance to other known β-lactamases; however, the sulbactam binding is found at two sites rich in positively charged residues, mainly at the L2 loop regions and the N-terminus. Using NMR spectroscopy, ITC and MD simulations, we map the ligand binding sites in UVI31 + providing atomic-level insights into its β-lactamase activity. Current study is the first report on β-lactamase activity of UVI31+, a BolA analogue, from C. reinhartii. Furthermore, our mutation studies reveal that the active site serine-55 is crucial for β-lactamase activity.

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Local Structures in Proteins from Microsecond Molecular Dynamics Simulations: 2. The Role of Symmetry in GTPase Binding and Dimer Formation.

The Rho GTPase binding domain of Plexin-B1 (RBD) prevails in solution as dimer. Under appropriate circumstances, it binds the small GTPase Rac1 to yield the complex RBD-Rac1. Here, we study RBD dimerization and complex formation from a symmetry-based perspective using data derived from 1 μs long MD simulations. The quantities investigated are the local potentials, u(MD), prevailing at the N-H sites of the protein. These potentials are statistical in character providing an empirical description of the local structure. To establish more methodical description, a method for approximating them by explicit functions, u(simulated), was developed in the preceding article in this journal issue. These functions are combinations of analytical Wigner functions, DL,K, belonging to the D2h point group. The D2h subgroups Ag and B2u are found to dominate u(simulated); the B1u subgroup contributes in some cases. The Ag (B2u) functions have axial or rhombic symmetry. For the first time, local potentials in proteins can be quantitatively characterized in terms of their strength (rhombicity) evaluated by axial Ag (rhombic Ag and B2u) contributions. Until now, the chain-segment [β3-L3-β4] and to some extent the α2-helix have been associated with GTPase binding. Here, we find that this process causes an increase (decrease) in the potential strength of β3 and β4 (the preceding L2 loop and the remote chain-segment [(α2-helix)-(α2/β5-turn)-(β5-strand)]), suggesting effects of counterbalancing and allostery. There is evidence for the L2 loop being associated with RBD-GTPase binding. Until now only the L4 loop has been associated with RBD dimerization. The latter process is found to cause an increase (decrease) in the potential strength and rhombicity of the L4 loop (the adjacent chain-segment [(α2-helix)-(α2/β5-turn)-(β5-strand)]), suggesting counterbalancing activity. On average, the RBD dimer features stronger local potentials than RBD-Rac1. The novel information inherent in these findings is mesoscopic in character. Prospects of interest include exploring relation to atomistic force-field parameters.

Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Discovery of aurones bearing two amine functionalities as SHIP2 inhibitors with insulin-sensitizing effect in rat myotubes.

Pharmacological inhibition of the SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) by small-molecule compounds presents an attractive approach to modulate insulin sensitivity. Few drug-like SHIP2 inhibitors have been discovered to date. A series of aurones incorporating key motifs from known SHIP2 inhibitors were synthesized and evaluated for SHIP2-inhibiting activity against a recombinant SHIP2 protein in vitro. Three aurones that inhibited SHIP2 at 15-50 μM were identified. These aurone inhibitors required two amine functionalities, one at ring A and a second at ring B for good inhibitory activity as exemplified by 12a. Mechanistically, molecular dynamics simulations revealed 12a to preferably bind to an allosteric site, restricting the motion of the flexible L4 loop required for SHIP2 phosphatase activity. Additionally, a basic piperidine moiety of 12a interacted with an aspartate residue proximal to the site. At 20-40 μM, 12a significantly enhanced glucose uptake in rat myotubes via increased Akt phosphorylation. 12a showed good permeability across the Caco-2 cell monolayer supporting the aurone chemotype as a new lead to develop drug-like, oral insulin sensitizers.

Molecular dynamics simulations at high temperatures of the Aeropyrum pernix L7Ae thermostable protein: Insight into the unfolding pathway

Most life forms on earth live at temperatures below 50 °C. Within these organisms are proteins that form the three-dimensional structures essential to their biological activity and function. However, some thermophilic life forms can resist higher temperatures and have corresponding adaptations to preserve protein function at these high temperatures. Among the structural factors responsible for this resistance of thermophilic proteins to high temperatures is the presence of additional hydrogen bonds in the thermophilic proteins, which means that the structure of the protein is more resistant to unfolding. Similarly, thermostable proteins are rich in structure-stabilizing salt bridges and/or disulfide bridges. In this context, we perform multiple replica molecular dynamics simulations at different temperatures on the Aeropyrum pernix (L7Ae) protein (from the crenarchaeal species A. pernix), known for its high melting temperature, and this in the aim to elucidate the structural factors responsible for its high thermostability. The results reveal that between the most sensitive regions of the protein to the increase of temperature are the loops L1, and L5, which surround the hydrophobic core region of the protein, besides the loop L9, and the C-terminal α5 region. This latter is the longer alpha helix of the protein secondary structure motifs and it is the first to be denaturated at 450 K, while the rest of the protein secondary structure motifs at this temperature were intact. The mechanism of unfolding that follows this protein at 550 K is similar to other thermophile proteins found in literature, with the opening of the loops that surround the hydrophobic core of the protein. So, the latter is completely exposed to the solvent, and partially denatured. The total denaturation process of the protein takes an average time of 40 ns to be achieved. Our investigation also shows that all the calculated salt bridges, with distances less than or equal to 6 A°, are on the periphery part of the protein, exposed to the solvent. However, the hydrophobic core of the protein is not involved in the formation of salt bridges, but rather with formation of some important hydrogen bondings that still persist even at 450 K. So, optimizing hydrogen bonding, near or within the core region, at high temperatures is a strategy that follows this thermostable protein to protect its hydrophobic core from denaturation, and ensure the thermal stability of the protein.

Design, synthesis, and biological evaluation of HDAC6 inhibitors targeting L1 loop and serine 531 residue

Histone deacetylases (HDACs) are a group of enzymes that remove acetyl groups from histones, leading to the silencing of genes. Targeting specific isoforms of HDACs has emerged as a promising approach for cancer therapy, as it can overcome drawbacks associated with pan-HDAC inhibitors. HDAC6 is a unique HDAC isoform that deacetylates non-histone proteins and is primarily located in the cytoplasm. It also has two catalytic domains and a zinc-finger ubiquitin binding domain (Zf-UBD) unlike other HDACs. HDAC6 plays a critical role in various cellular processes, including cell motility, protein degradation, cell proliferation, and transcription. Hence, the deregulation of HDAC6 is associated with various malignancies. In this study, we report the design and synthesis of a series of HDAC6 inhibitors. We evaluated the synthesized compounds by HDAC enzyme assay and identified that compound 8g exhibited an IC50 value of 21 nM and 40-fold selective activity towards HDAC6. We also assessed the effect of compound 8g on various cell lines and determined its ability to increase protein acetylation levels by Western blotting. Furthermore, the increased acetylation of α-tubulin resulted in microtubule polymerization and changes in cell morphology. Our molecular docking study supported these findings by demonstrating that compound 8g binds well to the catalytic pocket via L1 loop of HDAC6 enzyme. Altogether, compound 8g represents a preferential HDAC6 inhibitor that could serve as a lead for the development of more potent and specific inhibitors.

Anillin-related Mid1 as an adaptive and multimodal contractile ring anchoring protein: A simulation study

Cytokinesis of animal and fungi cells depends crucially on the anillin scaffold proteins. Fission yeast anillin-related Mid1 anchors cytokinetic ring precursor nodes to the membrane. However, it is unclear if both of its Pleckstrin Homology (PH) and C2 C-terminal domains bind to the membrane as monomers or dimers, and if one domain plays a dominant role. We studied Mid1 membrane binding with all-atom molecular dynamics near a membrane with yeast-like lipid composition. In simulations with the full C terminal region started away from the membrane, Mid1 binds through the disordered L3 loop of C2 in a vertical orientation, with the PH away from the membrane. However, a configuration with both C2 and PH initially bound to the membrane remains associated with the membrane. Simulations of C2-PH dimers show extensive asymmetric membrane contacts. These multiple modes of binding may reflect Mid1's multiple interactions with membranes, node proteins, and ability to sustain mechanical forces.

Antibiotic Charge Profile Determines the Extent of L3 Dynamics in OmpF: An Expedited Passage for Molecules with a Positive Charge.

Efficient permeation into Gram-negative bacterial cells is a much-desired property in the design of antibacterial agents. The goal is to arrive at one or more chemical modifications of molecules that improve their uptake into the cell while maintaining a good binding affinity to the intracellular target. Previously, we proposed a mechanistic rationale for the fast permeation of bulky antibiotics that involves induced conformational dynamics in the constriction loop L3 of the OmpF channel. This flexibility is caused by the perturbation of a hydrogen bond network stabilizing the L3 loop due to the strong interactions of the positively charged moiety on the antibiotic with the residues of the L3 loop. In the present work, we examine how differences in the charge profile of antibiotic molecules can affect the permeation process, in particular, the L3 dynamics. To this end, we have performed all-atom molecular dynamics simulations to study the permeation process of molecules with differences in the net charge through the Escherichia coli OmpF channel. The results from these simulations suggest that a positively charged moiety on the antibiotic is responsible for strong interactions with the negatively charged residues of the L3 loop, promoting conformational dynamics in the L3 loop. In contrast, antibiotics without a positively charged moiety are unable to initiate such a dynamic response in the L3 loop. This distinct behavior of the L3 loop in the presence of molecules with different charge characteristics provides a plausible mechanism whereby large molecules with an appropriate charge distribution can leverage an L3 dynamic-dependent pathway to permeate efficiently. The results are relevant to the structure-based design of molecules with improved uptake properties achieved through systematic chemical modifications that effectively engage the L3 loop.

Role of Conformational Dynamics of Sulfotransferases SULT1A1 and SULT1A3 in Substrate Specificity

Sulfotransferases (SULTs) are phase II metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3′-Phosphoadenosine 5′-Phosphosulfate (PAPS) to a wide variety of endogenous compounds, drugs and natural products. Although SULT1A1 and SULT1A3 share 93% identity, SULT1A1, the most abundant SULT isoform in humans, exhibits a broad substrate range with specificity for small phenolic compounds, while SULT1A3 displays a high affinity toward monoamine neurotransmitters like dopamine. To elucidate the factors determining the substrate specificity of the SULT1 isoenzymes, we studied the dynamic behavior and structural specificities of SULT1A1 and SULT1A3 by using molecular dynamics (MD) simulations and ensemble docking of common and specific substrates of the two isoforms. Our results demonstrated that while SULT1A1 exhibits a relatively rigid structure by showing lower conformational flexibility except for the lip (loop L1), the loop L2 and the cap (L3) of SULT1A3 are extremely flexible. We identified protein residues strongly involved in the recognition of different substrates for the two isoforms. Our analyses indicated that being more specific and highly flexible, the structure of SULT1A3 has particularities in the binding site, which are crucial for its substrate selectivity.