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Results for “ATP binding”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
NASA NTRS2001

Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

Non-NASA Center↗
DOE OSTI2023

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

Ion channels and the transduction of light signals

Studies of biological light-sensing mechanisms are revealing important roles for ion channels. Photosensory transduction in plants is no exception. In this article, the evidence that ion channels perform such signal-transducing functions in the complex array of mechanisms that bring about plant photomorphogenesis will be reviewed and discussed. The examples selected for discussion range from light-gradient detection in unicellular algae to the photocontrol of stem growth in Arabidopsis. Also included is some discussion of the technical aspects of studies that combine electrophysiology and photobiology.

NASA Discipline Plant Biology↗
NASA NTRS2000

Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

NASA Discipline Plant Biology↗
NASA NTRS1997

Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

Non-NASA Center↗
DOE OSTI2022

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with viral genome replication. Here in this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can initially bind in a comparatively stabilized state to the viral RdRp active site, as it primarily forms base stacking with the template uracil nucleotide (nt +1), which under freely fluctuations supports a low free energy barrier of the RDV-TP insertion (~1.5 kcal mol -1 ). In comparison, the corresponding natural substrate ATP binds initially to the RdRp active site in Watson–Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (~2.6 kcal mol -1 ), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be specifically stabilized by motif C-S759, S682 (near motif B) with the base, and motif G-K500 with the template backbone. Although the RDV-TP insertion can be hindered by motif F-R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif A-D623, respectively.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Crystal structure of the 4-hydroxybutyryl-CoA synthetase (ADP-forming) from nitrosopumilus maritimus

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl-CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Expression and characterization of spore coat Cot$\mathrm{H}$ kinases from the cellulosomes of anaerobic fungi ( Neocallimastigomycetes )

Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg 2+ binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2 -/- and Spdya -/- phenotypes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a distinctive mode of engaging poly(ADP-ribose)

PARP13/ZAP (zinc-finger antiviral protein) acts against multiple viruses by promoting degradation of viral mRNA. PARP13 has four N-terminal zinc (Zn) fingers that bind CG-rich nucleotide sequences, a C-terminal ADP ribosyltransferase fold, and a central region with a fifth Zn finger and tandem WWE domains. The central PARP13 region, ZnF5-WWE1-WWE2, is implicated in binding poly(ADP-ribose); however, there are limited insights into its structure and function. We present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and in complex with ATP. The crystal structures and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose) chains. The composite ZnF5-WWE1-WWE2 structure forms an extended surface to engage ADP-ribose chains, representing a distinctive mode of recognition that provides a framework for investigating the impact of poly(ADP-ribose) on PARP13 function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Conformational motions and ligand-binding underlying gating and regulation in IP 3 R channel

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP 3 and Ca 2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP 3 R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP 3 , Ca 2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP 3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP 3 R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte

We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

NASA Discipline Cardiopulmonary↗
DOE OSTI2020

Nitrogenase Bioelectrocatalysis: ATP-Independent Ammonia Production Using a Redox Polymer/MoFe Protein System

Nitrogenase is the only biological catalyst that is understood to be able to convert nitrogen gas to ammonia. In microorganisms, the MoFe catalytic protein of nitrogenase is reduced by a transient Fe protein binding and separate hydrolysis of ATP. Yet, the requirement of 16 ATP molecules by the Fe protein for the 8 electron transfer is an energy-intense caveat to the enzymatic synthesis of NH 3 and is challenging from an electrochemical perspective. Thus, we report the redox polymer-based ATP-free mediated electron-transfer system of MoFe nitrogenase using cobaltocene-functionalized poly(allylamine) (Cc-PAA), which is able to reduce the MoFe nitrogenase directly with a low redox potential of -0.58 V vs SHE. An efficient immobilization of MoFe nitrogenase via Cc-PAA allowed for the bioelectrocatalytic reduction of N 3 – , NO 2 – , and N 2 to NH 3 . Bulk bioelectrosynthetic experiments produced 7 ± 2 and 30 ± 5 nmol of NH 3 from NO 2 – and N 3 – reduction for 30 min, respectively. In addition, biosynthetic N 2 reduction to NH 3 was confirmed by 15 N 2 labeling experiments with NMR analysis. This mediated electron-transfer approach of the immobilized nitrogenase using the Cc-PAA redox polymer provides a valuable technological basis for scale-up and industrial uses in the future of bioelectrosynthesis.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
NASA NTRS2000

Models of the Protocellular Structures, Functions and Evolution

In the absence of extinct or extant record of protocells, the most direct way to test our understanding of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids: First, a very large population of candidate molecules is generated using a random synthetic approach. Next, the small numbers of molecules that can accomplish the desired task are selected. These molecules are next vastly multiplied using the polymerase chain reaction. A mutagenic approach, in which the sequences of selected molecules are randomly altered, can yield further improvements in performance or alterations of specificities. Unfortunately, the catalytic potential of nucleic acids is rather limited. Proteins are more catalytically capable but cannot be directly amplified. In the new technique, this problem is circumvented by covalently linking each protein of the initial, diverse, pool to the RNA sequence that codes for it. Then, selection is performed on the proteins, but the nucleic acids are replicated. To date, we have obtained "a proof of concept" by evolving simple, novel proteins capable of selectively binding adenosine tri-phosphate (ATP). Our next goal is to create an enzyme that can phosphorylate amino acids and another to catalyze the formation of peptide bonds in the absence of nucleic acid templates. This latter reaction does not take place in contemporary cells. once developed, these enzymes will be encapsulated in liposomes so that they will function in a simulated cellular environment. To provide a continuous energy supply, usually needed to activate the substrates, an energy transduction complex which generates ATP from adenosine diphosphate, inorganic phosphate and light will be used. This system, consisting of two modern proteins, ATP synthase and bacteriorhodopsin, has already been built and shown to work efficiently. By coupling chemical synthesis to such a system, it will be possible to drive chemical reactions by light if only the substrates for these reactions are supplied.

Pohorille, Andrew↗
DOE OSTI2025

Pyrrole‐Imine Macrocycle: Self‐Organizing Cross‐Reactive Anion Receptor and Sensor

Self-organizing macrocyclic receptor-sensors for phosphorus oxyanions, phosphates, and phosphonates comprising imine moieties were prepared by condensation of dipyrrolylmethane dicarbaldehyde with diethylene triamine. The incorporation of flexible ethylene moieties endows the macrocycle with unprecedented flexibility and ability to accommodate numerous phosphorus oxyanions from orthophosphate to large anions such as ATP or phosphonate glyphosate. The anion binding was elucidated by NMR titrations, low-temperature NMR, and NOESY NMR. The incorporation of dansyl fluorophore enables sensing of anions using the fluorescence signal, whereas the changes in fluorescence intensity, width of the fluorescence band, and position of the maxima are analyte-specific and useful in recognition and identification of eleven different P-oxyanions in water. The affinity (K assoc ) for Na + salts was H 2 PO 4 − ≈ Methylphosphonate > H 2 P 2 O 7 2− > Phenylphosphonate- > Glyphosate 2− > AMP 2− > ADP 2− > ATP 2− . Interestingly, phosphonates, including methylphosphonate and glyphosate anions, were also found to display a strong affinity (K assoc ∼10 6 M −1 ) while halides, nitrate, carbonates, or hydrogen sulfate did not show a significant affinity. The determined fluorescence spectral parameters were used to classify the 12 analytes (11 anions and water) using Linear Discriminant Analysis (LDA). Quantification was performed using LDA and Support Vector Machine (SVM), and the phosphonate concentrations in unknown samples were determined with an error of 3.5% or lower.

anions↗
DOE OSTI2022

SPARC: Structural properties associated with residue constraints

SPARC facilitates the generation of plausible hypotheses regarding underlying biochemical mechanisms by structurally characterizing protein sequence constraints. Such constraints appear as residues co-conserved in functionally related subgroups, as subtle pairwise correlations (i.e., direct couplings), and as correlations among these sequence features or with structural features. SPARC performs three types of analyses. First, based on pairwise sequence correlations, it estimates the biological relevance of alternative conformations and of homomeric contacts, as illustrated here for death domains. Second, it estimates the statistical significance of the correspondence between directly coupled residue pairs and interactions at heterodimeric interfaces. Third, given molecular dynamics simulated structures, it characterizes interactions among constrained residues or between such residues and ligands that: (a) are stably maintained during the simulation; (b) undergo correlated formation and/or disruption of interactions with other constrained residues; or (c) switch between alternative interactions. We illustrate this for two homohexameric complexes: the bacterial enhancer binding protein (bEBP) NtrC1, which activates transcription by remodeling RNA polymerase (RNAP) containing σ 54 , and for DnaB helicase, which opens DNA at the bacterial replication fork. Based on the NtrC1 analysis, we hypothesize possible mechanisms for inhibiting ATP hydrolysis until ADP is released from an adjacent subunit and for coupling ATP hydrolysis to restructuring of σ 54 binding loops. Based on the DnaB analysis, we hypothesize that DnaB ‘grabs’ ssDNA by flipping every fourth base and inserting it into cavities between subunits and that flipping of a DnaB-specific glutamine residue triggers ATP hydrolysis.

97 MATHEMATICS AND COMPUTING↗