Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.
ABSTRACT: Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations.
Project description:How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle.
Project description:The effect of surface chemistry on the adsorption characteristics of a fibronectin fragment (FNIII8?10) was investigated using fully atomistic molecular dynamics simulations. Model surfaces were constructed to replicate self-assembled monolayers terminated with methyl, hydroxyl, amine, and carboxyl moieties. It was found that adsorption of FNIII8?10 on charged surfaces is rapid, specific, and driven by electrostatic interactions, and that the anchoring residues are either polar uncharged or of opposing charge to that of the targeted surfaces. On charged surfaces the presence of a strongly bound layer of water molecules and ions hinders FNIII8?10 adsorption. In contrast, adsorption kinetics on uncharged surfaces are slow and non-specific, as they are driven by van der Waals interactions, and the anchoring residues are polar uncharged. Due to existence of a positively charged area around its cell-binding region, FNIII8?10 is available for subsequent cell binding when adsorbed on a positively charged surface, but not when adsorbed on a negatively charged surface. On uncharged surfaces, the availability of the fibronectin fragment's cell-binding region is not clearly distinguished because adsorption is much less specific.
Project description:During the development of the central nervous system (CNS), oligodendrocytes wrap their plasma membrane around axons to form a multilayered stack of tightly attached membranes. Although intracellular myelin compaction and the role of myelin basic protein has been investigated, the forces that mediate the close interaction of myelin membranes at their external surfaces are poorly understood. Such extensive bilayer-bilayer interactions are usually prevented by repulsive forces generated by the glycocalyx, a dense and confluent layer of large and negatively charged oligosaccharides. Here we investigate the molecular mechanisms underlying myelin adhesion and compaction in the CNS. We revisit the role of the proteolipid protein and analyze the contribution of oligosaccharides using cellular assays, biophysical tools, and transgenic mice. We observe that differentiation of oligodendrocytes is accompanied by a striking down-regulation of components of their glycocalyx. Both in vitro and in vivo experiments indicate that the adhesive properties of the proteolipid protein, along with the reduction of sialic acid residues from the cell surface, orchestrate myelin membrane adhesion and compaction in the CNS. We suggest that loss of electrostatic cell-surface repulsion uncovers weak and unspecific attractive forces in the bilayer that bring the extracellular surfaces of a membrane into close contact over long distances.
Project description:Sarcoplasmic reticulum Ca(2+)-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca(2+) transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K(+) is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.
Project description:In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na(+)-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na(+)-driven motor than in the MotA-FliG interactions in the H(+)-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na(+)-driven motor seem to be more complex than those previously proposed in the H(+)-driven motor.
Project description:The speed at which a molecular motor operates is critically important for the survival of a virus or an organism but very little is known about the underlying mechanisms. Tailed bacteriophage T4 employs one of the fastest and most powerful packaging motors, a pentamer of gp17 that translocates DNA at a rate of up to ?2000-bp/s. We hypothesize, guided by structural and genetic analyses, that a unique hydrophobic environment in the catalytic space of gp17-adenosine triphosphatase (ATPase) determines the rate at which the 'lytic water' molecule is activated and OH- nucleophile is generated, in turn determining the speed of the motor. We tested this hypothesis by identifying two hydrophobic amino acids, M195 and F259, in the catalytic space of gp17-ATPase that are in a position to modulate motor speed. Combinatorial mutagenesis demonstrated that hydrophobic substitutions were tolerated but polar or charged substitutions resulted in null or cold-sensitive/small-plaque phenotypes. Quantitative biochemical and single-molecule analyses showed that the mutant motors exhibited 1.8- to 2.5-fold lower rate of ATP hydrolysis, 2.5- to 4.5-fold lower DNA packaging velocity, and required an activator protein, gp16 for rapid firing of ATPases. These studies uncover a speed control mechanism that might allow selection of motors with optimal performance for organisms' survival.
Project description:Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal-terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279-316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase-portal orientation fits better to a proposed "DNA crunching" compression packaging motor and to portal determined DNA headful cutting.
Project description:Tailed bacteriophages use powerful molecular motors to package the viral genome into a preformed capsid. Packaging at a rate of up to ?2000 bp/s and generating a power density twice that of an automobile engine, the phage T4 motor is the fastest and most powerful reported to date. Central to DNA packaging are dynamic interactions among the packaging components, capsid (gp23), portal (gp20), motor (gp17, large "terminase"), and regulator (gp16, small terminase), leading to precise orchestration of the packaging process, but the mechanisms are poorly understood. Here we analyzed the interactions between small and large terminases of T4-related phages. Our results show that the gp17 packaging ATPase is maximally stimulated by homologous, but not heterologous, gp16. Multiple interaction sites are identified in both gp16 and gp17. The specificity determinants in gp16 are clustered in the diverged N- and C-terminal domains (regions I-III). Swapping of diverged region(s), such as replacing C-terminal RB49 region III with that of T4, switched ATPase stimulation specificity. Two specificity regions, amino acids 37-52 and 290-315, are identified in or near the gp17-ATPase "transmission" subdomain II. gp16 binding at these sites might cause a conformational change positioning the ATPase-coupling residues into the catalytic pocket, triggering ATP hydrolysis. These results lead to a model in which multiple weak interactions between motor and regulator allow dynamic assembly and disassembly of various packaging complexes, depending on the functional state of the packaging machine. This might be a general mechanism for regulation of the phage packaging machine and other complex molecular machines.
Project description:Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.
Project description:Organelle transport in eukaryotes employs both microtubule and actin tracks to deliver cargo effectively to their destinations, but the question of how the two systems cooperate is still largely unanswered. Recently, in vitro studies revealed that the actin-based processive motor myosin V also binds to, and diffuses along microtubules. This biophysical trick enables cells to exploit both tracks for the same transport process without switching motors. The detailed mechanisms underlying this behavior remain to be solved. By means of single molecule Total Internal Reflection Microscopy (TIRFM), we show here that electrostatic tethering between the positively charged loop 2 and the negatively charged C-terminal E-hooks of microtubules is dispensable. Furthermore, our data indicate that in addition to charge-charge interactions, other interaction forces such as non-ionic attraction might account for myosin V diffusion. These findings provide evidence for a novel way of myosin tethering to microtubules that does not interfere with other E-hook-dependent processes.