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1.  FAN1 Activity on Asymmetric Repair Intermediates Is Mediated by an Atypical Monomeric Virus-type Replication-Repair Nuclease Domain 
Cell Reports  2014;8(1):84-93.
Summary
FAN1 is a structure-selective DNA repair nuclease with 5′ flap endonuclease activity, involved in the repair of interstrand DNA crosslinks. It is the only eukaryotic protein with a virus-type replication-repair nuclease (“VRR-Nuc”) “module” that commonly occurs as a standalone domain in many bacteria and viruses. Crystal structures of three representatives show that they structurally resemble Holliday junction resolvases (HJRs), are dimeric in solution, and are able to cleave symmetric four-way junctions. In contrast, FAN1 orthologs are monomeric and cleave 5′ flap structures in vitro, but not Holliday junctions. Modeling of the VRR-Nuc domain of FAN1 reveals that it has an insertion, which packs against the dimerization interface observed in the structures of the viral/bacterial VRR-Nuc proteins. We propose that these additional structural elements in FAN1 prevent dimerization and bias specificity toward flap structures.
Graphical Abstract
Highlights
•Bacterial proteins comprising solely a VRR-Nuc domain are dimeric in solution•Bacterial VRR-Nuc domains act as Holliday-junction-resolving enzymes•A conserved helical insertion in the FAN1 VRR-Nuc domain prevents dimerization•FAN1 monomers cannot cleave Holliday junctions and instead cleave 5′ flap DNA
The Fanconi anemia pathway is responsible for clearing DNA interstrand crosslinks that block DNA replication and transcription leading to genome instability. Here, Pennell et al. characterize the Fanconi-anemia-associated nuclease FAN1, comparing the structure and activity of its catalytic VRR-Nuc domain with prokaryotic examples. FAN1 is monomeric with 5′ flap specificity, whereas prokaryotic VRR-Nuc domains are dimeric Holliday-junction-resolving enzymes. FAN1 is proposed to contain a conserved helical insertion blocking dimer formation and consequently restricting substrate specificity.
doi:10.1016/j.celrep.2014.06.001
PMCID: PMC4103454  PMID: 24981866
2.  H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis 
PLoS Pathogens  2012;8(9):e1002914.
Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, KM values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (KI) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.
Author Summary
Recently, a pandemic A/H1N1 influenza virus was isolated from an immune compromised patient with a novel antiviral resistance pattern to the neuraminidase inhibitor class of drugs. This virus had an amino acid change in the viral neuraminidase enzyme; an isoleucine at position 223 was substituted by an arginine (I223R). Patients infected with such a virus leave physicians with reduced antiviral treatment options, since pandemic viruses are naturally resistant to the other class of antivirals, the adamantanes. Previously, we have shown that this mutant virus retains its potential to cause disease and may still be able to spread in the human population.
Here we used enzyme kinetic measurements and crystal structures of the I223R mutant neuraminidase to determine the resistance mechanism of this amino acid change. We found that the I223R change results in shrinkage of the active site of the enzyme. As a result, binding of the neuraminidase inhibitors is affected. In addition, we found that the active site of our pandemic neuraminidase structure, crystallized in the absence of inhibitor, has an extra cavity (150-cavity) adjacent to the active site. Our study could aid in the development of novel inhibitors designed to target the 150-cavity and active site of the enzyme.
doi:10.1371/journal.ppat.1002914
PMCID: PMC3447749  PMID: 23028314
3.  Crystallization of SHARPIN using an automated two-dimensional grid screen for optimization 
The expression, purification and crystallization of an N-terminal fragment of SHARPIN are reported. Diffraction-quality crystals were obtained using a two-dimensional grid-screen seeding technique.
An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli, purified and crystallized. Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen. The crystals belonged to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 61.55, c = 222.81 Å. Complete data sets were collected from native and selenomethionine-substituted protein crystals at 100 K to 2.6 and 2.0 Å resolution, respectively.
doi:10.1107/S1744309112022208
PMCID: PMC3388930  PMID: 22750873
SHARPIN; seeding
4.  Structural Analysis of SHARPIN, a Subunit of a Large Multi-protein E3 Ubiquitin Ligase, Reveals a Novel Dimerization Function for the Pleckstrin Homology Superfold* 
The Journal of Biological Chemistry  2012;287(25):20823-20829.
Background: SHARPIN is a subunit of the E3 ligase complex LUBAC and the gene that is mutated in chronic proliferative dermatitis mice.
Results: The N-terminal portion of SHARPIN adopts the PH superfold and mediates homodimerization.
Conclusion: The PH superfold can be used as a protein dimerization module.
Significance: This study highlights the versatility of the PH superfold and its function as a protein interaction module.
SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.
doi:10.1074/jbc.M112.359547
PMCID: PMC3375506  PMID: 22549881
Protein Structure; Protein-Protein Interactions; Signal Transduction; Ubiquitin Ligase; Ubiquitination; X-ray Crystallography
5.  The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator 
Nucleic Acids Research  2012;40(9):3913-3928.
Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail’ dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.
doi:10.1093/nar/gkr1300
PMCID: PMC3351161  PMID: 22234878
6.  ADP Regulates SNF1, the Saccharomyces cerevisiae Homolog of AMP-Activated Protein Kinase 
Cell metabolism  2011;14(5):707-714.
SUMMARY
The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.
doi:10.1016/j.cmet.2011.09.009
PMCID: PMC3241989  PMID: 22019086
7.  ADP Regulates SNF1, the Saccharomyces cerevisiae Homolog of AMP-Activated Protein Kinase 
Cell Metabolism  2011;14(5):707-714.
Summary
The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.
Graphical Abstract
Highlights
► ADP regulates SNF1 by protecting against dephosphorylation of Thr210 ► Snf4 binds two molecules of adenine nucleotide ► NADH binds to site 4 within Snf4, but does not compete with ADP protection ► Mechanism for ADP protection is conserved between SNF1 and AMPK
doi:10.1016/j.cmet.2011.09.009
PMCID: PMC3241989  PMID: 22019086
8.  Structure of Mammalian AMPK and its regulation by ADP 
Nature  2011;472(7342):230-233.
The heterotrimeric AMP-activated protein kinase (AMPK) plays a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy producing pathways and inhibits energy consuming processes1. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer 2,3,4,5,6. AMPK is converted from an inactive to catalytically competent form by phosphorylation of the activation loop within the kinase domain7; AMP binding to the γ regulatory domain promotes phosphorylation by the upstream kinase8, protects the enzyme against dephosphorylation as well as causing allosteric activation9. We show here that ADP binding to just one of the two exchangeable AXP binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active AMPK displays significantly tighter binding to ADP than to Mg.ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg.ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. It shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity (Supplementary Fig. 1).
doi:10.1038/nature09932
PMCID: PMC3078618  PMID: 21399626
9.  A super-modular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA-damage 
Cell  2009;139(1):100-111.
SUMMARY
The Mre11/Rad50/Nbs1 (MRN) protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3Å crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA and BRCT-repeat domains structurally coalesce. We demonstrate that di-phosphorylated pSer-Asp-pThr-Asp-like motifs, recently identified as multi-copy docking sites within human Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we identify similar phospho-motifs within Ctp1, the fission yeast orthologue of the human tumor-suppressor, CtIP, and show that their interactions with the Nbs1 FHA domain are necessary for Ctp1-dependent resistance to DNA-damage. Finally, we establish that human Nbs1 interactions with Mdc1 can occur through both its FHA and BRCT-repeat domains, suggesting how their structural and functional inter-dependence may underpin Nbs1 adaptor functions in the DNA-damage response.
doi:10.1016/j.cell.2009.07.043
PMCID: PMC2900601  PMID: 19804756
10.  Structural basis of AMPK regulation by small molecule activators 
Nature Communications  2013;4:3017.
AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.
Small molecule activators of the energy sensing kinase AMPK are promising candidates as therapies for metabolic disease. Xiao et al. present the crystal structure of AMPK in complex with a small molecule activator, and show that the drug stabilizes interaction between the catalytic and carbohydrate-binding domains.
doi:10.1038/ncomms4017
PMCID: PMC3905731  PMID: 24352254
11.  The malaria parasite egress protease SUB1 is a calcium-dependent redox switch subtilisin 
Nature Communications  2014;5:3726.
Malaria is caused by a protozoan parasite that replicates within an intraerythrocytic parasitophorous vacuole. Release (egress) of malaria merozoites from the host erythrocyte is a highly regulated and calcium-dependent event that is critical for disease progression. Minutes before egress, an essential parasite serine protease called SUB1 is discharged into the parasitophorous vacuole, where it proteolytically processes a subset of parasite proteins that play indispensable roles in egress and invasion. Here we report the first crystallographic structure of Plasmodium falciparum SUB1 at 2.25 Å, in complex with its cognate prodomain. The structure highlights the basis of the calcium dependence of SUB1, as well as its unusual requirement for interactions with substrate residues on both prime and non-prime sides of the scissile bond. Importantly, the structure also reveals the presence of a solvent-exposed redox-sensitive disulphide bridge, unique among the subtilisin family, that likely acts as a regulator of protease activity in the parasite.
In the malarial parasitophorous vacuole, the serine protease SUB1 processes parasite proteins that are required for release from host cells and invasion. Here, the authors report the first crystallographic structure of SUB1 in complex with its cognate prodomain revealing its substrate interactions and providing insight into its regulation.
doi:10.1038/ncomms4726
PMCID: PMC4024747  PMID: 24785947

Results 1-11 (11)