Pathological variants in Trk-fused gene (TFG) have been implicated in a variety of neurodegenerative conditions. In particular, mutations within its amino-terminal PB1 domain have been suggested to cause hereditary spastic paraplegia (HSP), resulting in progressive lower limb spasticity and weakness. The structural basis for this effect is unknown. Here, we combine X-ray crystallography and cryo-electron microscopy to determine a structural model of TFG, demonstrating the mechanism by which it forms octameric ring complexes. A network of electrostatic and hydrophobic interactions defines the interface between protomers. Moreover, we show that mutations identified previously in HSP patients disrupt this interface, destabilizing octamers, which ultimately leads to axonopathy. Surprisingly, the impacts of these variants are not equivalent in vivo, highlighting the existence of multiple, distinct mechanisms by which TFG mutations contribute to neurodegenerative disease.

Fumarase C (FumC) catalyzes the reversible conversion of fumarate to S ‐malate. Previous structural investigations within the superfamily have reported a dynamic structural segment, termed the SS Loop. To date, active‐site asymmetry has raised the question of how SS Loop placement affects participation of key residues during the reaction. Herein, we report structural and kinetic analyses from Escherichia coli FumC variants to understand the contribution of SS Loop residues S318, K324, and N326. High‐resolution X‐ray crystallographic results reveal three distinct FumC active‐site conformations; disordered‐open, ordered‐open, and the newly discovered ordered‐closed. Surprisingly, each SS Loop variant has unaffected Michaelis constants coupled to reductions in turnover number. Based upon our structural and functional analyses, we propose structural and catalytic roles for each of the aforementioned residues.

The citric acid cycle is a vital pathway for virulence of facultative bacterial pathogens. Two of these bacteria, Salmonella enterica and Escherichia coli, pose a major threat for foodborne illness. Here we present the characterization of an enzyme in the citric acid cycle as targets for the development of novel antibacterial agents. Fumarase C catalyzes the reversible reaction of fumarate to malate. Fumarate conversion to malate in a necessary piece of the CAC cycle. S. enterica has been shown to lose virulence when the genes encoding its fumarase C enzyme (SeFumC) are knocked out, making this protein a potential drug target against S. enterica infections. To gain kinetic and structural knowledge of SeFumC, we have cloned the gene encoding FumC into a plasmid for overexpression. We have purified SeFumC‐His6 using immobilized metal affinity chromatography. We have baseline values of Michaelis‐Menten kinetic parameters for the SeFumC. We are investigating the stability of SeFumC folding using circular dichroism. To facilitate structural analysis, we have identified a condition that promotes crystallization of SeFumC. Characterizing the kinetic and structural features of SeFumC will aid in the potential development of future antibiotics that target the citric acid cycle in bacterial pathogens.Support or Funding InformationUWL Eagle Scholars Apprenticeship Program

Fumarase belongs to a larger superfamily of enzymes including aspartase, adenylosuccinate lyase, arginosuccinate lyase, and 3-carboxy-cis, cis-muconate lactonizing enzyme. Each enzyme member shares a common quaternary structure and harbors a flexible active site loop, termed the SS Loop. The SS Loop lies within the fumarase superfamily signature sequence 317GS318SxMxK324xN326xxPxE331 and plays a fundamental role during catalysis. Recently, human fumarase deficiencies have been linked to renal cell carcinoma and benign uterine tumor formation. In this study we site-selectively altered SS Loop residues serine 318, lysine 324, asparagine 326 and glutamic acid 331 to alanine. The fumarase C SS Loop variants were characterized functionally and structurally. Functionally, steady-state kinetic experiments were conducted in the classic Krebs Cycle sense (fumarate to S-malate) and in the reductive biosynthetic direction (S-malate to fumarate). Structurally, the single-site SS Loop variants were analyzed via circular dichroism and x-ray crystallography. The steady-state kinetic results report minimal effect on the S-malate and fumarate Km values, while reducing kcat significantly. We have placed the kcat reduction into two categories: (1) diminished charge-relay influence and (2) SS Loop immobility. Structurally, the SS Loop single-site alanine variants have similar thermal and chemical denaturation profiles. Thus, we suggest the overall homotetrameric quaternary structure, a requisite for a functional multi-subunit active site, is intact. X-ray crystallographic results for both the serine 318 to alanine (S318A) and lysine 324 to alanine (K324A) variants report a newly discovered fumarase C active site architecture. The SS Loop in both the S318A and K324A active sites undertakes two disparate conformations leading to two unique active sites. These two active sites are non-crystallographically related and undertake ordered-open and ordered-closed conformations. Thus, alteration of serine 318 and lysine 324 has facilitated crystallographic order within the SS Loop region. Ordering and closure of the S318A SS Loop has created an electrostatic gate between the multi-subunit active site and an auxiliary single-subunit citrate binding site. Adaptation within the K324A crystal structure includes additionally ordered active site water molecules. Effectively, the newly observed water molecules re-establish electrostatic interactions between serine 98, threonine 100, asparagine 141, histidine 188 and the previously observed active site water. Our structural and functional results further establish the importance of SS Loop conformational mobility within the fumarase superfamily. S318A SS Loop and domain 3 movement lead to closure of the fumarase C active site. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DcrB is an 18 kDa lipoprotein that contains a single domain of unknown function. DcrB is found within Enterobacteriaceae, a family of Gram-negative bacteria which includes pathogens that can cause food-borne illness and hospital-acquired infections. In Salmonella enterica serovar Typhimurium, DcrB is up-regulated by conditions that promote the production of known virulence factors. We determined the structure of a truncated form of DcrB from Salmonella to 1.92 angstrom resolution by X-ray crystallography. This truncated form, DcrB Delta 37, contains the entire domain of unknown function but lacks the lipoprotein signal sequence (residues 1-20) as well as residues 21-37. The DcrB Delta 37 monomer contains the Mog1p/PsbP-like fold, which is found in functionally diverse proteins in mammals, yeast, plants, and cyanobacteria. Interestingly, DcrB Delta 37 crystallized as a domain swapped homodimer in which the N-terminal beta-hairpin extends from one protomer to interact with the core of the second protomer. This domain-swapping indicates that the N-terminal portion of the Mog1p/PsbP-like fold likely has conformational flexibility. Overall, our results provide the first example of an enterobacterial protein that contains the Mog1p/PsbP-like fold and expands knowledge of the structural and phylogenetic diversity of Mog1p/PsbP-like proteins.

Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved 'DNA replication restart' pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT).

β‐helix structures are a unique structural domain typified by the helical arrangement of parallel β‐strands. The uniqueness of the β‐helix structure can be further deconstructed into individual β‐circuits, where each β‐circuit describes a single revolution of three contiguous parallel β‐strands. Inherently, the cooperative formation of consecutive β‐circuits is driven by on‐edge main chain hydrogen bonds between adjacent parallel β‐strands. This cooperatively folded domain affords both structural (1) stability and (2) malleability. Therefore, the β‐helix structure has been recruited as a robust structural domain for numerous bacterial virulence factors, including adhesins, hemolysins, and heme‐binding proteins.A truncated version of hemolysin A (HpmA265) from Proteus mirabilis has been implemented as a model to probe the stability and malleability of the β‐helix structure. Hemolysin A belongs to the two‐partner secretion pathway, which is the most widely distributed protein secretion system within gram‐negative bacteria. Hemolysin A is secreted and concomitantly activated via hemolysin B, its cognate TPS outer membrane β‐barrel component. Structurally, HpmA265 harbors the putative TPS domain, but lacks the functional pore‐forming domain. Recent equilibrium unfolding studies have dissected the larger TPS domain into three sequentially folding subdomains, termed the polar core, non‐polar core, and carboxy‐terminal subdomains. Moreover, polar core subdomain was proposed to serve as a template to couple the vectorial alignment of parallel β‐strands with nucleotide independent protein secretion across the outer membrane.In order to probe key interactions as associated with β‐helix stability and malleability, a series of HpmA265 variants were prepared and analyzed functionally and structurally. The first set of variants was constructed to investigate the importance of a previously observed buried hydrogen bond between Gln125 and Tyr134 within the polar core subdomain. Additionally, the importance of the on‐edge hydrogen bonds was investigated through the limited proteolysis of HpmA265. Replacement of Gln125 and Tyr134 was conducted singly and in tandem with alanine, serine or phenylalanine. Each of the polar core variants was analyzed structurally via equilibrium unfolding and x‐ray crystallographic studies. Interestingly, the double alanine replacement at Gln125 and Tyr134 establishes a 200 Å3 cavity buried within the HpmA265 β‐helix structure. Moreover, the double serine replacement establishes an unprecedented inner core hydrogen bond network involving seven well‐resolved water molecules. The high resolution crystallographic results allowed visualization and placement of each water molecule buried within the HpmA265 β‐helix structure, while the equilibrium unfolding studies supported destabilization within the polar core subdomain. The limited proteolysis study has established a previously unobserved HpmA265 dimerization interface facilitated via newly exposed on‐edge main chain hydrogen bond partners. This newly observed dimerization interface extends cooperative β‐circuit formation via the implementation of exposed parallel β‐strands donated by adjacent monomers. Collectively, the results support the β‐helix as a stable and malleable structural domain.Support or Funding InformationThis research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB‐12002) and the National Institute of General Medical Sciences (AGM‐12006). The research was supported in part by National Science Foundation Grant: MCB1050435 (TW) and a University Wisconsin – La Crosse Faculty Research Grant (TW).

Living cells require magnesium ions for many fundamental biochemical processes. Certain pathogenic bacteria can thrive in environments with limiting magnesium, and the ability to adapt to magnesium limitation can be critical for bacterial virulence. We have used genetic and biochemical approaches to gain insight into how Salmonella enterica , a major bacterial cause of food‐borne illness, adapts to low magnesium. Using a genetic screen in a Salmonella mutant defective for adaptation to low magnesium, we identified a periplasmic lipoprotein protein that promotes growth in low magnesium. This Salmonella protein, which has a monomeric molecular weight of 19.7 kDa, contains a periplasmic localization sequence followed by a single domain of unknown function that is conserved among enterobacteria. To elucidate its function, we have purified and crystallized this protein as well as obtained atomic‐resolution X‐ray diffraction data. Circular dichroism spectroscopy indicates that the protein consists predominantly of beta‐sheet secondary structure, and the diffraction data indicate there is likely more than one protein monomer in the asymmetric unit. Overall, our results demonstrate a physiological function in magnesium ion homeostasis for a previously uncharacterized periplasmic lipoprotein in Salmonella , and we have obtained the first atomic‐resolution structural data for a member of this family of proteins. These discoveries will provide insight into molecular mechanisms of bacterial adaptation to magnesium limitation. Support or Funding Information This research is supported by University of Wisconsin‐La Crosse Undergraduate Research and Creativity Grants (DR, TD), the McNair Scholars Program at UW‐La Crosse (DR), and a University of Wisconsin‐La Crosse Faculty Research Grant (JM). The authors would also like to thank Professor James Keck (University Wisconsin, Department of Biomolecular Chemistry) for graciously allowing access to the Advanced Photon Source. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB‐12002) and the National Institute of General Medical Sciences (AGM‐12006).

Wild-type and variant forms of HpmA265 (truncated hemolysin A) fromProteus mirabilisreveal a right-handed, parallel β-helix capped and flanked by segments of antiparallel β-strands. The low-salt crystal structures form a dimeric structureviathe implementation of on-edge main-chain hydrogen bonds donated by residues 243–263 of adjacent monomers. Surprisingly, in the high-salt structures of two variants, Y134A and Q125A-Y134A, a new dimeric interface is formedviamain-chain hydrogen bonds donated by residues 203–215 of adjacent monomers, and a previously unobserved tetramer is formed. In addition, an eight-stranded antiparallel β-sheet is formed from the flap regions of crystallographically related monomers in the high-salt structures. This new interface is possible owing to additional proteolysis of these variants after Tyr240. The interface formed in the high-salt crystal forms of hemolysin A variants may mimic the on-edge β-strand positioning used in template-assisted hemolytic activity.

The divalent magnesium cation (Mg 2+ ) is imperative to the survival of all living organisms on earth. It stabilizes different macromolecular and cellular structures, and plays a vital role in a vast number of essential biochemical reactions. Due to its significance, many organisms have evolved complex biochemical systems to adapt to varying environmental levels of Mg 2+ . Additionally, the ability to adapt to environments with limiting Mg 2+ is required by many bacteria to cause disease in humans. Gaining insight into how bacteria maintain homeostasis when in magnesium deficient environments will provide critical knowledge in finding new treatments for antibiotic resistant bacteria. Through a genetic screen, we have identified a novel periplasmic lipoprotein of unknown structure and biochemical function that rescues growth of a mutant strain of Salmonella enterica that is unable to respond to magnesium deficient environments. Interestingly, this lipoprotein has a single domain of unknown function that is conserved among a large number of disease causing enteric bacteria. To elucidate the structural characteristics of the protein, circular dichroism (CD) and X‐ray crystallography techniques are being used. CD experiments have revealed that the protein is composed primarily of beta‐sheet secondary structure, which confirms the secondary structure predicted by homology modeling. Thermal denaturations analyzed by CD reveal a single state reversible transition with a melting temperature of approximately 71 °C. Additionally, we are progressing towards obtaining the first high resolution atomic structure of this protein. We have developed reproducible crystal conditions for the native protein and have successfully obtained atomic resolution X‐ray diffraction data which suggests more than one monomer per asymmetric unit. Initial attempts at molecular replacement with this data have so far been unsuccessful. Selenomethionine (SeMet) was incorporated into the native protein, and has also been successfully crystallized. Obtaining X‐ray diffraction data on these crystals will provide the phasing information necessary to solve the first atomic resolution structure of this protein. The structure will ultimately provide us with knowledge of this protein's role in magnesium homeostasis within Salmonella. Support or Funding Information This work was supported by the University of Wisconsin‐ La Crosse Undergraduate Research and Creativity grant (DR), McNair Scholars program (DR), and a University of Wisconsin‐ La Crosse Faculty Research grant (JM). The authors would also like to thank Professor James Keck (University Wisconsin, Department of Biomolecular Chemistry) for allowing access to the Advanced Photon Source. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB‐12002) and the National Institute of General Medical Sciences (AGM‐12006).

Maintenance and faithful transmission of genomic information depends on the efficient execution of numerous DNA replication, recombination, and repair pathways. Many of the enzymes that catalyze steps within these pathways require access to sequence information that is buried in the interior of the DNA double helix, which makes DNA unwinding an essential cellular reaction. The unwinding process is mediated by specialized molecular motors called DNA helicases that couple the chemical energy derived from nucleoside triphosphate hydrolysis to the otherwise non-spontaneous unwinding reaction. An impressive number of high-resolution helicase structures are now available that, together with equally important mechanistic studies, have begun to define the features that allow this class of enzymes to function as molecular motors. In this review, we explore the structural features within DNA helicases that are used to bind and unwind DNA. We focus in particular on aromatic-rich loops that allow some helicases to couple single-stranded DNA binding to ATP hydrolysis and wedge/pin elements that provide mechanical tools for DNA strand separation when connected to translocating motor domains.

Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.

DNA replication restart is an essential genome maintenance process by which replication complexes (replisomes) are reloaded onto abandoned DNA replication forks. In Escherichia coli and related bacteria this process is orchestrated by the PriA DNA helicase, which binds to replication forks in a structure‐specific manner. PriA also binds to single‐stranded DNA‐binding protein (SSB), a protein that coats the lagging‐strand template at DNA replication forks. Complex formation with SSB stimulates PriA DNA unwinding DNA and, as we show here, this interaction is required for PriA remodeling of SSB‐DNA nucleoprotein complexes. Proteins that interact with SSB typically do so by binding directly to the evolutionarily conserved amphipathic C‐terminal tail of SSB (SSB‐CT). Although previous studies indicated that PriA also binds to SSB via the SSB‐CT, a lack of structural information on PriA has left the SSB binding site on PriA unknown. We determined the 4 Å resolution structure of PriA bound to the SSB‐CT. The electrostatic characteristics of the PriA SSB‐Ct binding site resemble those of other known SSB‐interacting proteins. Single‐molecule FRET studies show that a PriA variant that has lost the ability to bind the SSB‐CT fails to modulate SSB‐DNA complexes. Our studies point to the importance of the PriA/SSB interaction in allowing PriA to reload replisomes at damaged replication forks where SSB is bound to the lagging‐strand template.

Approximately one disruption in DNA replication occur every cell cycle in bacteria leading to partially duplicated chromosomes. Since unfinished replication can result in genome instability and cell death, bacteria need a mechanism to reload the replication machinery onto the genome. Known as the replication restart primosome (RRP), several proteins function to reload the replicative helicase onto abandoned replication forks, restarting DNA replication. PriA is the most conserved member of the RRP, initiating the dominant replication restart pathway. A helicase, PriA remodels collapsed forks and serves as a platform for binding of other primosomal proteins. The Madison West High School Students Modeling a Research Topic (SMART) Team modeled PriA using 3D printing technology. PriA is a multi‐domain protein and residues important for DNA binding, ATP hydrolysis, and helicase activity are modeled. Since DNA replication restart pathways are essential in preserving genomic integrity and cell viability in bacteria, studies of PriA offer an approach to developing novel antibacterial compounds. Supported by a grant from NIH‐CTSA.

The treatment of bacterial diseases has become a serious problem in clinical medicine in large part because of antibiotic resistance. One way bacteria have increased resistance to drugs is the overexpression of multidrug efflux pumps, exemplified by AcrAB‐TolC, a group of three proteins that span both the inner and outer membranes of E. coli. AcrAB‐TolC transports a wide range of foreign compounds and a broad spectrum of antibiotics. AcrB forms a homo‐trimeric structure that uses cyclical conformational changes powered by ATP to transport foreign compounds into the TolC pore which eventually expels the compounds from the bacteria. The role of AcrA is less defined, perhaps providing structural support to AcrB. The Madison West High School SMART Team (Students Modeling A Research Topic) modeled the interactions between AcrB and associated proteins in the E. coli pump using 3D printing technology to further study the mechanisms of bacterial drug efflux, as well as gain a better understanding of other drug efflux transport mechanisms, such as that used by P‐glycoprotein, which is important in cancer biology and human resistance to chemotherapeutics. The study of the detailed structures of AcrAB‐TolC may be of paramount importance in the development of novel pharmaceuticals against bacterial infections. Supported by grants from NIH‐SEPA and NIH‐CTSA.

DNA replication is a complex molecular process that requires the coordination of almost a dozen different proteins. Central to this molecular dance is DNA polymerase, which synthesizes new DNA strands in the 5′ to 3′ direction. While DNA is replicated continuously on the leading strand, the anti‐parallel nature of DNA coupled with the unidirectional nature of DNA polymerases poses an inherent obstacle in synthesis of the lagging strand. To overcome this challenge, organisms synthesize the lagging strand discontinuously as Okazaki fragments. Initiation of each Okazaki fragment requires an enzyme called primase, which is an RNA polymerase that synthesizes short RNA primers that can be extended by DNA polymerase.. The Madison West High School Students Modeling a Research Topic (SMART) team has modeled the core catalytic domain of DNA primase from Escherichia coli using Jmol and rapid prototyping in order to gain a better understanding of the mechanisms of DNA replication. Supported by grants from theHoward Hughes Medical Institute and NIH‐NCRR‐SEPA

DNA replication is a vital process in all organisms and understanding the fundamental biochemical interactions that drive replication is essential. Single‐stranded DNA‐binding (SSB) proteins form an important component of the replication machinery that facilitates the transfer of RNA primers from the enzyme primase to the replicative polymerase. This activity occurs throughout lagging‐strand DNA replication. The crystal structure of the E. coli χψ subunit of the DNA polymerase III holoenzyme bound to its interaction site on SSB has been determined. The Madison West High School Students Modeling a Research Topic (SMART) Team, in collaboration with the Milwaukee School of Engineering, has modeled this interaction using 3D Rapid Prototyping Technology to gain insights into the physical interactions that drive DNA replication. The SMART team program allows students to experience the scientific process beyond the textbook by investigating the experimental methodology of structural biology and takes students out of the classroom and into the laboratory. Supported by grants from the Howard Hughes Medical Institute and NIH‐NCRR‐SEPA.

Each year, nearly 40% of cancer patients in the U.S. die of the disease and more than 50% of tumors contain mutations of the TP53 gene encoding tumor suppressor protein p53. p53 functions in cell cycle control, senescence, and apoptosis in response to external stress. In normal cells, p53 binds the protein MDM2, promoting p53 degradation. Once activated by an environmental stress (DNA damage, oncogenes, or hypoxia), p53 releases MDM2, and, in turn, binds DNA. p53 induces the expression of p21, a protein that arrests cell division. p53 mutants are unable to activate p21, resulting in uncontrolled cell division and ultimately cancer.. Individuals with only one functional copy of TP53 will most likely develop Li‐Fraumeni Syndrome characterized by multiple tumor development in early adulthood. Pathogens such as human papillomavirus (HPV) can produce proteins that inactivate p53, leading to a higher incidence of cervical cancer. The Madison West High School SMART (Students Modeling a Research Topic) Team in collaboration with MSOE have built accurate three‐dimensional models of human p53 bound to DNA using 3D printing technology. By modeling p53, we hope to better understand how mutations in the protein lead to cancer as well as elucidating better mechanisms of cancer treatment. Supported by grants from the Howard Hughes Medical Institute, NIH‐NCRR‐SEPA and NIH T32 GM07215‐33.