Assembly of phage Mu transpososomes: Cooperative transitions assisted by protein and DNA scaffolds

AbstractTransposition of phage Mu takes place within higher order protein-DNA complexes called transpososomes. These complexes contain the two Mu genome ends synapsed by a tetramer of Mu transposase (MuA). Transpososome assembly is tightly controlled by multiple protein and DNA sequence cofactors. We find that assembly can occur through two distinct pathways. One previously described pathway depends on an enhancer-like sequence element, the internal activation sequence (IAS). The second pathway depends on a MuB protein-target DNA complex. For both pathways, all four MuA monomers in the tetramer need to interact with an assembly-assisting element, either the IAS or MuB. However, once assembled, not all MuA monomers within the transpososome need to interact with MuB to capture MuB-bound target DNA. The multiple layers of control likely are used in vivo to ensure efficient rounds of DNA replication when needed, while minimizing unwanted transposition products

Segregation of molecules at cell division reveals native protein localization

We introduce a non-intrusive method exploiting post-division single-cell variability to validate protein localization. The results show that Clp proteases, widely reported to form biologically relevant foci, are in fact uniformly distributed inside Escherichia coli cells, and that many commonly used fluorescent proteins (FPs) cause severe mislocalization when fused to homo-oligomers. Re-tagging five other reportedly foci-forming proteins with the most monomeric FP tested suggests the foci were caused by the FPs

Characterization of a Specificity Factor for an AAA+ ATPase Assembly of SspB Dimers with ssrA-Tagged Proteins and the ClpX Hexamer

AbstractSspB, a specificity factor for the ATP-dependent ClpXP protease, stimulates proteolysis of protein substrates bearing the ssrA degradation tag. The SspB protein is shown here to form a stable homodimer with two independent binding sites for ssrA-tagged proteins or peptides. SspB by itself binds to ClpX and stimulates the ATPase activity of this enzyme. In the presence of ATPγS, a ternary complex of SspB, GFP-ssrA, and the ClpX ATPase was sufficiently stable to isolate by gel-filtration or ion-exchange chromatography. This complex consists of one SspB dimer, two molecules of GFP-ssrA, and one ClpX hexamer. SspB dimers do not commit bound substrates to ClpXP degradation but increase the affinity and cooperativity of binding of ssrA-tagged substrates to ClpX, facilitating enhanced degradation at low substrate concentrations

Deciphering the Roles of Multicomponent Recognition Signals by the AAA+ Unfoldase ClpX

ATP-dependent protein remodeling and unfolding enzymes are key participants in protein metabolism in all cells. How these often-destructive enzymes specifically recognize target protein complexes is poorly understood. Here, we use the well-studied AAA + unfoldase-substrate pair, Escherichia coli ClpX and MuA transposase, to address how these powerful enzymes recognize target protein complexes. We demonstrate that the final transposition product, which is a DNA-bound tetramer of MuA, is preferentially recognized over the monomeric apo-protein through its multivalent display of ClpX recognition tags. The important peptide tags include one at the C-terminus (“C-tag”) that binds the ClpX pore and a second one (enhancement or “E-tag”) that binds the ClpX N-terminal domain. We construct a chimeric protein to interrogate subunit-specific contributions of these tags. Efficient remodeling of MuA tetramers requires ClpX to contact a minimum of three tags (one C-tag and two or more E-tags), and that these tags are contributed by different subunits within the tetramer. The individual recognition peptides bind ClpX weakly (K[subscript D] > 70 μM) but impart a high-affinity interaction (K[subscript D] ~ 1.0 μM) when combined in the MuA tetramer. When the weak C-tag signal is replaced with a stronger recognition tag, the E-tags become unnecessary and ClpX's preference for the complex over MuA monomers is eliminated. Additionally, because the spatial orientation of the tags is predicted to change during the final step of transposition, this recognition strategy suggests how AAA + unfoldases specifically distinguish the completed “end-stage” form of a particular complex for the ideal biological outcome.National Institutes of Health (U.S.) (Grants GM-49224 and AI-16892)National Institutes of Health (U.S.) (NIH Pre-Doctoral Training Grant T32GM007287

AAA+ protease-adaptor structures reveal altered conformations and ring specialization

AbstractClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here we present high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze the protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated, tucked away from the channel and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies show additional structures and functions for key AAA+ elements. Pore-loop tucking may be used broadly by AAA+ unfoldases, for example, during enzyme pausing/unloading.</jats:p

Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism. Keywords: heme biosynthesis; porphyria; ALAS; protein unfoldases; AAA+ ATPaseNational Institutes of Health (U.S.) (Grant F32 DK095726)National Institutes of Health (U.S.) (Grant R01 GM049224

A Threat Assessment and Security Analysis of the Three Sports Facilities of Indiana University-Purdue University, Indianapolis NCAA Softball Fields, Carroll Stadium, and the IU Natatorium

This research report provides a security assessment of the Softball Fields, Carroll Stadium, and the Natatorium Complex at Indiana University-Purdue University Indianapolis (IUPUI). The purpose of this report is to prevent and mitigate harm to visitors and these facilities which resulting from human-made or natural disasters. Research is guided by the hypothesis that these facilities- due to their respective importance, locations, and attendance patterns are in harm’s way; and that certain strategies of prevention, protection, and mitigation coupled with effective preparedness, response, and recovery can lessen risk, improve security and provide A THREAT ASSESSMENT AND SECURITY ANALYSIS 5 added resilience. Further, “harm’s way” is considered to be either a natural disaster or a human-made disaster, accident, active provocation, or act of terrorism. Methods of analysis include applied research; predominantly utilizing qualitative data with some quantitative investigation. Results of this assessment illustrate that these venues possess numerous vulnerabilities to both natural and human-made threats that if exposed, could result in serious consequences. The two most likely natural hazards identified include straight-line winds and tornadoes. Further, the most likely human threats to these facilities arise from a potential terrorist vehicle attack (TVA) and an active shooter. This project also identifies a specific need for additional planning to prevent an IED or VBIED attack on the Natatorium. Common themes from the attached three case studies reveal that given theses vulnerabilities, the following safety and security adjustments are recommended: Surveillance equipment Metal detectors Security bollards or other temporary barriers Evacuation routes and shelter in place plans Special event security procedures Weather related technology and protocols Staff training for emergency situation

A pilot Internet "Value of Health" Panel: recruitment, participation and compliance

Objectives To pilot using a panel of members of the public to provide preference data via the Internet Methods A stratified random sample of members of the general public was recruited and familiarised with the standard gamble procedure using an Internet based tool. Health states were perdiodically presented in "sets" corresponding to different conditions, during the study. The following were described: Recruitment (proportion of people approached who were trained); Participation (a) the proportion of people trained who provided any preferences and (b) the proportion of panel members who contributed to each "set" of values; and Compliance (the proportion, per participant, of preference tasks which were completed). The influence of covariates on these outcomes was investigated using univariate and multivariate analyses. Results A panel of 112 people was recruited. 23% of those approached (n = 5,320) responded to the invitation, and 24% of respondents (n = 1,215) were willing to participate (net = 5.5%). However, eventual recruitment rates, following training, were low (2.1% of those approached). Recruitment from areas of high socioeconomic deprivation and among ethnic minority communities was low. Eighteen sets of health state descriptions were considered over 14 months. 74% of panel members carried out at least one valuation task. People from areas of higher socioeconomic deprivation and unmarried people were less likely to participate. An average of 41% of panel members expressed preferences on each set of descriptions. Compliance ranged from 3% to 100%. Conclusion It is feasible to establish a panel of members of the general public to express preferences on a wide range of health state descriptions using the Internet, although differential recruitment and attrition are important challenges. Particular attention to recruitment and retention in areas of high socioeconomic deprivation and among ethnic minority communities is necessary. Nevertheless, the panel approach to preference measurement using the Internet offers the potential to provide specific utility data in a responsive manner for use in economic evaluations and to address some of the outstanding methodological uncertainties in this field

Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX

The hexameric AAA+ ring of Escherichia coli ClpX, an ATP-dependent machine for protein unfolding and translocation, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+-ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U↔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+-ring activity.National Institutes of Health (U.S.) (Grant GM-101988)Massachusetts Institute of Technology (Poitras Predoctoral Fellowship