Viral Packaging ATPases Utilize a Glutamate Switch to Couple ATPase Activity and DNA Translocation [preprint]

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in apo, ATP-bound, and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an inactive to an active pose upon ATP binding, and that a residue assigned as the glutamate switch is necessary for regulating the transition. Further, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental data. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the predicted structural coupling from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway in viral DNA packaging motors that ensures coordination between chemical and mechanical events involved in viral DNA packaging

Atomistic Mechanism of Force Generation, Translocation, and Coordination in a Viral Genome Packaging Motor [preprint]

Double-stranded DNA viruses package their genomes into pre-assembled protein capsids using virally-encoded ATPase ring motors. While several structures of isolated monomers (subunits) from these motors have been determined, they provide little insight into how subunits within a functional ring coordinate their activities to efficiently generate force and translocate DNA. Here we describe the first atomic-resolution structure of a functional ring form of a viral DNA packaging motor and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Crystal structures of the pentameric ATPase ring from bacteriophage asccφ28 show that each subunit consists of a canonical N-terminal ASCE ATPase domain connected to a ‘vestigial’ nuclease domain by a small lid subdomain. The lid subdomain closes over the ATPase active site and engages in extensive interactions with a neighboring subunit such that several important catalytic residues are positioned to function in trans. The pore of the ring is lined with several positively charged residues that can interact with DNA. Simulations of the ATPase ring in various nucleotide-bound states provide information about how the motor coordinates sequential nucleotide binding, hydrolysis, and exchange around the ring. Simulations also predict that the ring adopts a helical structure to track DNA, consistent with recent cryo-EM reconstruction of the φ29 packaging ATPase. Based on these results, an atomistic model of viral DNA packaging is proposed wherein DNA translocation is powered by stepwise helical-to-planar ring transitions that are tightly coordinated by ATP binding, hydrolysis, and release

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccphi28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous phi29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases

Low-Cost Water Pump Design/Testing to Serve Rural Villages (Semester Unknown) IPRO 323: Low-Cost Water Pump DesignTesting to Serve Rural Villages IPRO 323 Project Plan Sp08

The objective of IRPO 323 is to find and optimize a system to deliver drinkable water to communities that do not have easy access to water. Mainly in locating a water source accessible to the region based on geographical limitations. From there, creation of a system to draw out the water in order to meet the demands of the people based on their lifestyle. The system has to be easy to maintain, fulfill the water requirement, and is cost effective in the life of the design. However, in order to fully understand the system and its limitations a test model will be used. In order to accomplish this project in a timely manner each individual will have to learn, develop, and apply interpersonal skills, research methods, and coordination. The set objectives, therefore, are:  Evaluate water sources and water demands of the target community  Design a system for this community  Research products and costs for system components  Create a test model to better understand the system componentsSponsorship: US Environmental Protection Agency, National Collegiate Inventors & Innovators Alliance (NCIIA) and Stuart GrantsDeliverable

Low-Cost Water Pump Design/Testing to Serve Rural Villages (Semester Unknown) IPRO 323: Low-Cost Water Pump DesignTesting to Serve Rural Villages IPRO 323 MidTerm Presentation Sp08

The objective of IRPO 323 is to find and optimize a system to deliver drinkable water to communities that do not have easy access to water. Mainly in locating a water source accessible to the region based on geographical limitations. From there, creation of a system to draw out the water in order to meet the demands of the people based on their lifestyle. The system has to be easy to maintain, fulfill the water requirement, and is cost effective in the life of the design. However, in order to fully understand the system and its limitations a test model will be used. In order to accomplish this project in a timely manner each individual will have to learn, develop, and apply interpersonal skills, research methods, and coordination. The set objectives, therefore, are:  Evaluate water sources and water demands of the target community  Design a system for this community  Research products and costs for system components  Create a test model to better understand the system componentsSponsorship: US Environmental Protection Agency, National Collegiate Inventors & Innovators Alliance (NCIIA) and Stuart GrantsDeliverable

Low-Cost Water Pump Design/Testing to Serve Rural Villages (Semester Unknown) IPRO 323

The objective of IRPO 323 is to find and optimize a system to deliver drinkable water to communities that do not have easy access to water. Mainly in locating a water source accessible to the region based on geographical limitations. From there, creation of a system to draw out the water in order to meet the demands of the people based on their lifestyle. The system has to be easy to maintain, fulfill the water requirement, and is cost effective in the life of the design. However, in order to fully understand the system and its limitations a test model will be used. In order to accomplish this project in a timely manner each individual will have to learn, develop, and apply interpersonal skills, research methods, and coordination. The set objectives, therefore, are:  Evaluate water sources and water demands of the target community  Design a system for this community  Research products and costs for system components  Create a test model to better understand the system componentsSponsorship: US Environmental Protection Agency, National Collegiate Inventors & Innovators Alliance (NCIIA) and Stuart GrantsDeliverable

Low-Cost Water Pump Design/Testing to Serve Rural Villages (Semester Unknown) IPRO 323: Low-Cost Water Pump DesignTesting to Serve Rural Villages IPRO 323 Final Presentation Sp08

The objective of IRPO 323 is to find and optimize a system to deliver drinkable water to communities that do not have easy access to water. Mainly in locating a water source accessible to the region based on geographical limitations. From there, creation of a system to draw out the water in order to meet the demands of the people based on their lifestyle. The system has to be easy to maintain, fulfill the water requirement, and is cost effective in the life of the design. However, in order to fully understand the system and its limitations a test model will be used. In order to accomplish this project in a timely manner each individual will have to learn, develop, and apply interpersonal skills, research methods, and coordination. The set objectives, therefore, are:  Evaluate water sources and water demands of the target community  Design a system for this community  Research products and costs for system components  Create a test model to better understand the system componentsSponsorship: US Environmental Protection Agency, National Collegiate Inventors & Innovators Alliance (NCIIA) and Stuart GrantsDeliverable

Low-Cost Water Pump Design/Testing to Serve Rural Villages (Semester Unknown) IPRO 323: Low-Cost Water Pump DesignTesting to Serve Rural Villages IPRO 323 Poster2 Sp08

The objective of IRPO 323 is to find and optimize a system to deliver drinkable water to communities that do not have easy access to water. Mainly in locating a water source accessible to the region based on geographical limitations. From there, creation of a system to draw out the water in order to meet the demands of the people based on their lifestyle. The system has to be easy to maintain, fulfill the water requirement, and is cost effective in the life of the design. However, in order to fully understand the system and its limitations a test model will be used. In order to accomplish this project in a timely manner each individual will have to learn, develop, and apply interpersonal skills, research methods, and coordination. The set objectives, therefore, are:  Evaluate water sources and water demands of the target community  Design a system for this community  Research products and costs for system components  Create a test model to better understand the system componentsSponsorship: US Environmental Protection Agency, National Collegiate Inventors & Innovators Alliance (NCIIA) and Stuart GrantsDeliverable

Low-Cost Water Pump Design/Testing to Serve Rural Villages (Semester Unknown) IPRO 323: Low-Cost Water Pump DesignTesting to Serve Rural Villages IPRO 323 Ethics Sp08

The objective of IRPO 323 is to find and optimize a system to deliver drinkable water to communities that do not have easy access to water. Mainly in locating a water source accessible to the region based on geographical limitations. From there, creation of a system to draw out the water in order to meet the demands of the people based on their lifestyle. The system has to be easy to maintain, fulfill the water requirement, and is cost effective in the life of the design. However, in order to fully understand the system and its limitations a test model will be used. In order to accomplish this project in a timely manner each individual will have to learn, develop, and apply interpersonal skills, research methods, and coordination. The set objectives, therefore, are:  Evaluate water sources and water demands of the target community  Design a system for this community  Research products and costs for system components  Create a test model to better understand the system componentsSponsorship: US Environmental Protection Agency, National Collegiate Inventors & Innovators Alliance (NCIIA) and Stuart GrantsDeliverable

Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif

Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >107-fold decrease in phage yield, but we identified nine mutants with partial activity. Several were cold-sensitive, suggesting that mobility of the residues is important. Single-molecule measurements showed that the partially active A175L exhibits a small reduction in motor velocity and increase in slipping, consistent with a slowed ATP binding transition, whereas G176S exhibits decreased slipping, consistent with an accelerated transition. All changes to the conserved D178, predicted to coordinate Mg2+•ATP, were lethal except conservative change D178E. Biochemical interrogation of the inactive D178N protein found no folding or assembly defects and near-normal endonuclease activity, but a ∼200-fold reduction in steady-state ATPase activity, a lag in the single-turnover ATPase time course, and no DNA packaging, consistent with a critical role in ATP-coupled DNA translocation. Molecular dynamics simulations of related enzymes suggest that the aspartate plays an important role in enhancing the catalytic activity of the motor by bridging the Walker motifs and precisely contributing its charged group to help polarize the bound nucleotide. Supporting this prediction, single-molecule measurements revealed that change D178E reduces motor velocity without increasing slipping, consistent with a slowed hydrolysis step. Our studies thus illuminate the mechanistic roles of Walker-B residues in ATP binding, hydrolysis, and DNA translocation by this powerful motor