Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases.

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

Effect of wearable exoskeleton on post-stroke gait: A systematic review and meta-analysis.

BACKGROUND: Wearable exoskeletons are a recently developed technology. OBJECTIVES: The present systematic review aimed to investigate the effect of a wearable exoskeleton on post-stroke walking by considering its use in a gait training system and simply as an orthosis assisting walking. METHODS: We systematically searched for randomised and quasi-randomised controlled trials in PubMed, Scopus, CINAHL and Embase databases from their earliest publication record to July 2021. We chose reports of trials investigating the effects of exoskeleton-assisted training or the effects of wearing an exoskeleton to assist walking. A meta-analysis was conducted to explore the benefits of the wearable exoskeleton on mobility capacity, walking speed, motor function, balance, endurance and activities of daily living. RESULTS: We included 13 studies (492 participants) comparing exoskeleton-assisted training with dose-matched conventional gait training. Studies addressing the effect of wearing a wearable exoskeleton were unavailable. As compared with conventional gait training at the end of the intervention, exoskeleton-assisted training was superior for walking speed (mean difference [MD] 0.13 m/s, 95% CI 0.05; 0.21) and balance (standardized MD [SMD] 0.3, 95% CI 0.07; 0.54). The subgroup with chronic stroke (i.e., > 6 months) presented the outcome favouring exoskeleton-assisted training regarding overall mobility capacity (SMD 0.37, 95% CI 0.04; 0.69). At the end of follow-up, exoskeleton-assisted training was superior to conventional gait training in overall mobility (SMD 0.45, 95% CI 0.07; 0.84) and endurance (MD 46.23 m, 95% CI 9.90; 82.56). CONCLUSIONS: Exoskeleton-assisted training was superior to dose-matched conventional gait training in several gait-related outcomes at the end of the intervention and follow-up in this systematic review and meta-analysis, which may support the use of exoskeleton-assisted training in the rehabilitation setting. Whether wearing versus not wearing a wearable exoskeleton is beneficial during walking remains unknown.

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.

Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair.

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

Decoding Cancer Variants of Unknown Significance for Helicase-Nuclease-RPA Complexes Orchestrating DNA Repair During Transcription and Replication.

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase-nuclease-RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary "driver" and "passenger" mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

GRB2 enforces homology-directed repair initiation by MRE11.

DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.

EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart.

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.

A collagen glucosyltransferase drives lung adenocarcinoma progression in mice.

Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.

The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning.

The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates Pol II to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in vitro in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.

Protein supplementation increases adaptations to endurance training: A systematic review and meta-analysis.

BACKGROUND: Trials that assessed the impact of protein supplementation on endurance training adaptations have reported conflicting findings. OBJECTIVE: To determine the impact of protein supplementation during chronic endurance training on aerobic capacity, body composition and exercise performance in healthy and clinical populations. DESIGN: A systematic database search was conducted for randomised controlled trials addressing the effects of protein supplementation during endurance training on aerobic capacity, body composition and exercise performance in PubMed, Embase, Web of Science, and CINAHL. Meta-analyses were performed to outline the overall effects of protein supplementation with all studies containing endurance training components. The effects of endurance training and add-on effects of protein supplementation were evaluated by the meta-analyses with endurance training-focused studies. RESULTS: Nineteen studies and 1162 participants contributed to the analyses. Compared with the control group, the protein supplementation group demonstrated greater improvements in aerobic capacity measured by mixed peak oxygen uptake (V̇O2peak) and peak workload power (Wpeak) (standardised mean difference [SMD] = 0.36, 95% confidence interval [CI]: 0.05 to 0.67), and V̇O2peak (mean difference [MD] = 0.89 mL‧kg-1‧min-1, 95% CI: 0.07 to 1.70); had a greater lean mass gain (MD = 0.32 kg, 95% CI: 0.07 to 0.58); and had a greater improvement in time trial performance (MD = -29.1s, 95% CI:-55.3 to -3.0). Secondary analyses showed that, in addition to the substantial improvement in V̇O2peak (MD = 3.67 mL‧kg-1‧min-1, 95% CI: 2.32 to 5.03) attributed to endurance training, protein supplementation provided an additional 26.4% gain in V̇O2peak (MD = 0.97 mL‧kg-1‧min-1, 95% CI: -0.03 to 1.97). CONCLUSION: Protein supplementation further increased aerobic capacity, stimulated lean mass gain, and improved time trial performance during chronic endurance training in healthy and clinical populations. PROSPERO REGISTRATION NUMBER: (CRD42020155239).

Envisioning how the prototypic molecular machine TFIIH functions in transcription initiation and DNA repair.

Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant molecular structures and develop possible mechanistic insights for the large dynamic TFIIH complex. TFIIH presents many puzzles involving how its two SF2 helicase family enzymes, XPB and XPD, function in transcription initiation and repair: how do they initiate transcription, detect and verify DNA damage, select the damaged strand for incision, coordinate repair with transcription and cell cycle through Cdk-activating-kinase (CAK) signaling, and result in very different specific human diseases associated with cancer, aging, and development from single missense mutations? By joining analyses of breakthrough cryo-electron microscopy (cryo-EM) structures and advanced computation with data from biochemistry and human genetics, we develop unified concepts and molecular level understanding for TFIIH functions with a focus on structural mechanisms. We provocatively consider that TFIIH may have first evolved from evolutionary pressure for TCR to resolve arrested transcription blocks to DNA replication and later added its key roles in transcription initiation and global DNA repair. We anticipate that this level of mechanistic information will have significant impact on thinking about TFIIH, laying a robust foundation suitable to develop new paradigms for DNA transcription initiation and repair along with insights into disease prevention, susceptibility, diagnosis and interventions.

Effects of weight reduction on the breast cancer-related lymphedema: A systematic review and meta-analysis.

BACKGROUND: Obesity has long been considered a risk factor for breast cancer-related lymphedema (BCRL), but the benefits of weight reduction in managing BCRL have not been clearly established. OBJECTIVE: To evaluate the beneficial effects of weight loss interventions (WLIs) on the reduction and prevention of BCRL. METHODS: We conducted a systematic review and meta-analysis by searching the PubMed, Scopus, and Embase databases from their earliest record to October 1st, 2019. We included randomized and non-randomized controlled trials involving adult patients with a history of breast cancer, that compared WLI groups with no-WLI groups, and provided quantitative measurements of lymphedema. RESULTS: Initial literature search yielded 461 nonduplicate records. After exclusion based on title, abstract, and full-text review, four randomized controlled trials involving 460 participants were included for quantitative analysis. Our meta-analysis revealed a significant between-group mean difference (MD) regarding the volume of affected arm (MD = 244.7 mL, 95% confidence interval [CI]: 145.3-344.0) and volume of unaffected arm (MD = 234.5 mL, 95% CI: 146.9-322.1). However, a nonsignificant between-group MD of -0.07% (95% CI: 1.22-1.08) was observed regarding the interlimb volume difference at the end of the WLIs. CONCLUSIONS: In patients with BCRL, WLIs are associated with decreased volume of the affected and unaffected arms but not with decreased severity of BCRL measured by interlimb difference in arm volume.

The structure of the periplasmic FlaG-FlaF complex and its essential role for archaellar swimming motility.

Motility structures are vital in all three domains of life. In Archaea, motility is mediated by the archaellum, a rotating type IV pilus-like structure that is a unique nanomachine for swimming motility in nature. Whereas periplasmic FlaF binds the surface layer (S-layer), the structure, assembly and roles of other periplasmic components remain enigmatic, limiting our knowledge of the archaellum's functional interactions. Here, we find that the periplasmic protein FlaG and the association with its paralogue FlaF are essential for archaellation and motility. Therefore, we determine the crystal structure of Sulfolobus acidocaldarius soluble FlaG (sFlaG), which reveals a ß-sandwich fold resembling the S-layer-interacting FlaF soluble domain (sFlaF). Furthermore, we solve the sFlaG2-sFlaF2 co-crystal structure, define its heterotetrameric complex in solution by small-angle X-ray scattering and find that mutations that disrupt the complex abolish motility. Interestingly, the sFlaF and sFlaG of Pyrococcus furiosus form a globular complex, whereas sFlaG alone forms a filament, indicating that FlaF can regulate FlaG filament assembly. Strikingly, Sulfolobus cells that lack the S-layer component bound by FlaF assemble archaella but cannot swim. These collective results support a model where a FlaG filament capped by a FlaG-FlaF complex anchors the archaellum to the S-layer to allow motility.

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation.

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

Author Correction: Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding.

In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to also include support from the National Institutes of Health grant T32GM008280 to Sarah Alvarado.

Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes.

[Fe-S] clusters are essential cofactors in all domains of life. They play many biological roles due to their unique abilities for electron transfer and conformational control. Yet, producing and analyzing Fe-S proteins can be difficult and even misleading if not done anaerobically. Due to unique redox properties of [Fe-S] clusters and their oxygen sensitivity, they pose multiple challenges and can lose enzymatic activity or cause their component proteins to be structurally disordered due to [Fe-S] cluster oxidation and loss in air. Here we highlight tested protocols and strategies enabling efficient and stable [Fe-S] protein production, purification, crystallization, X-ray diffraction data collection, and structure determination. From multiple high-resolution anaerobic crystal structures, we furthermore analyze exemplary data defining [Fe-S] clusters, substrate entry, and product exit for the functional oxidation states of type II molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) enzymes. Notably, these enzymes perform electron shuttling between quinone pools and specific substrates to catalyze respiratory metabolism. The identified structure-activity relationships for this enzyme class have broad implications germane to perchlorate environments on Earth and Mars extending to an alternative mechanism underlying metabolic origins for the evolution of the oxygen atmosphere. Integrated structural analyses of type II Mo-bisMGD enzymes unveil novel distinctive shared molecular mechanisms for dynamic control of substrate entry and product release gated by hydrophobic residues. Collective findings support a prototypic model for type II Mo-bisMGD enzymes including insights for a fundamental molecular mechanistic understanding of selectivity and regulation by a conformationally gated channel with general implications for [Fe-S] cluster respiratory enzymes.

Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding.

Collagen lysyl hydroxylases (LH1-3) are Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus. Loop residues in the double-stranded ß-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe2+. The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe2+-binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase Km and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer.

The ATPase Motor Turns for Type IV Pilus Assembly.

In this issue of Structure, Mancl et al. (2016) elucidate the crystal structure of the PilB ATPase domain in complex with ATPγS and unveil how ATP binding and hydrolysis coordinates conformational change. Their results reveal a distinct symmetric rotary mechanism for ATP hydrolysis to power bacterial pilus assembly.

Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 µm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.