Boguslaw Nocek

A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR‐associated protein that is common to all CRISPR‐containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single‐stranded and branched DNAs including Holliday junctions, replication forks and 5′‐flaps. The crystal structure of YgbT and site‐directed mutagenesis have revealed the potential active site. Genome‐wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR–Cas system have a function in DNA repair.

Nuclease Activity of the Human SAMHD1 Protein Implicated in the Aicardi-Goutières Syndrome and HIV-1 Restriction

Background: The human SAMHD1 protein has dNTP triphosphatase activity and is involved in HIV-1 restriction and autoimmune syndrome. Results: Purified SAMHD1 exhibits nuclease activity against single-stranded DNA and RNA. Conclusion: The nuclease activity of SAMHD1 is associated with its HD domain. Significance: Identification of nuclease activity in SAMHD1 provides novel insight into the mechanisms of HIV-1 restriction and regulation of autoimmune response. The human HD domain protein SAMHD1 is implicated in the Aicardi-Goutières autoimmune syndrome and in the restriction of HIV-1 replication in myeloid cells. Recently, this protein has been shown to possess dNTP triphosphatase activity, which is proposed to inhibit HIV-1 replication and the autoimmune response by hydrolyzing cellular dNTPs. Here, we show that the purified full-length human SAMHD1 protein also possesses metal-dependent 3′→5′ exonuclease activity against single-stranded DNAs and RNAs in vitro. In double-stranded substrates, this protein preferentially cleaved 3′-overhangs and RNA in blunt-ended DNA/RNA duplexes. Full-length SAMHD1 also exhibited strong DNA and RNA binding to substrates with complex secondary structures. Both nuclease and dNTP triphosphatase activities of SAMHD1 are associated with its HD domain, but the SAM domain is required for maximal activity and nucleic acid binding. The nuclease activity of SAMHD1 could represent an additional mechanism contributing to HIV-1 restriction and suppression of the autoimmune response through direct cleavage of viral and endogenous nucleic acids. In addition, we demonstrated the presence of dGTP triphosphohydrolase and nuclease activities in several microbial HD domain proteins, suggesting that these proteins might contribute to antiviral defense in prokaryotes.

Large-scale evaluation of protein reductive methylation for improving protein crystallization

Large-scale evaluation of protein reductive methylation for improving protein crystallization

Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer α/β/α sandwich fold with an α-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE -Encoded N -succinyl-L,L-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapEs are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.

Differential stabilities and sequence-dependent base pair opening dynamics of watson-crick base pairs with 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxylcytosine.

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson–Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5′-CG-3′ sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5′-T8X9G10-3′ sequence of the DDD, were compared. The presence of 5caC at the X9 base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A5:T8, whereas 5caC did not. At the oxidized base pair G4:X9, 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C3:G10. No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G4:X9; each favored Watson–Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N4 exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.

Crystal structure of aminopeptidase N from human pathogen Neisseria meningitidis

Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and peptides for maturation, activation, or degradation, and therefore are involved in numerous biological processes.1,2 They are broadly distributed throughout all kingdoms of life and are found in subcellular organelles, cytoplasm, and in membrane-bound fractions.3 Many aminopeptidases use a set of conserved residues within a structural scaffold to form an active site capable of binding either one or two divalent metal ions that aid catalysis. Zn+2, Co+2, and Mn+2 are being the most common metals found in the active site.4–8 One of more extensively studied members of the aminopeptidase family is aminopeptidase N (APN) [alternative names: alanine aminopeptidase; aminopeptidase M; microsomal aminopeptidase; GP150; CD13; (EC 3.4.11.2)]. The APN sequence family is large and broadly distributed and includes members found in bacteria and eukaryotes, including plants and mammals. PsiBlast search identified over 1000 APN family members. Typically APNs are monomeric or homodimeric. In higher eukaryotes these enzymes are expressed in many tissues, with the highest level found in the intestinal and kidney brush border membranes, brain, lung, blood vessels, and primary cultures of fibroblasts. The sequence analysis indicates that aminopeptidase N is a member of the M1 family of the MA clan of peptidases, also termed gluzincins.5 The amino acid sequence fingerprints of the M1 family of zinc-metallopeptidases are the HEXXH(X18)E (a zinc binding motif) and GXMEN (an exopeptidase motif).9 Prominent members of this family include mammalian membrane-bound aminopeptidases [P-LAP, aminopeptidase A (APA), thyrotropin-releasing hormone degrading enzyme (TRHDE)], cytosolic proteins [puromycin-sensitive aminopeptidase (PSA) and leukotriene A4 hydrolase (LTA4H)], and secretory proteins such as [adipocyte-derived leucine aminopeptidase (A-LAP) and aminopeptidase B (APB)].5,9 The APNs catalyze liberation of N-terminal amino acids from a broad spectrum of substrates including small peptides, amide, or arylamide. The N-terminal residue is a preferably neutral or basic amino acid, although it has been reported that an intact XPro dipeptide was released when the terminal hydrophobic residue was followed by a prolyl residue.10 The diversity of function that APNs play depends on their location and source tissue. Some APNs have been used commercially, such as the APN from Lactococcus lactis, which has been used in the food industry.11 Aminopeptidases N are also present in many pathogenic bacteria and represent potential drug targets.9 In this article, we report the crystal structure of APN from N. meningitides at 2.05-A resolution.

Cleavable C-terminal His-tag vectors for structure determination

High-throughput structural genomics projects seek to delineate protein structure space by determining the structure of representatives of all major protein families. Generally this is accomplished by processing numerous proteins through standardized protocols, for the most part involving purification of N-terminally His-tagged proteins. Often proteins that fail this approach are abandoned, but in many cases further effort is warranted because of a protein’s intrinsic value. In addition, failure often occurs relatively far into the path to structure determination, and many failed proteins passed the first critical step, expression as a soluble protein. Salvage pathways seek to recoup the investment in this subset of failed proteins through alternative cloning, nested truncations, chemical modification, mutagenesis, screening buffers, ligands and modifying processing steps. To this end we have developed a series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-terminal tags. In an initial set of 16 proteins that failed with an N-terminal appendage, structures were obtained for C-terminally tagged derivatives of five proteins, including an example for which several alternative salvaging steps had failed. The new vectors allow appending C-terminal His6-tag and His6- and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases.

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS

Background: Haloacid dehalogenase (HAD)-like hydrolases represent the largest superfamily of phosphatases. Results: Biochemical, structural, and evolutionary studies of the 10 uncharacterized soluble HADs from Saccharomyces cerevisiae provided insight into their substrates, active sites, and evolution. Conclusion: Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Significance: Our work contributes to a better understanding of an important model organism. The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members.

Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

Significance There are many differences in the sequences of ketosynthase (KS) domains from the well-studied type I polyketide synthases (PKSs) and the more recently discovered acyltransferase (AT)-less type I PKSs. The AT-less type I PKSs generate polyketides with a high degree of structural diversity, which stems from their evolution by horizontal gene transfer. In comparison, canonical type I PKSs evolve by gene duplication. The seven structures of AT-less type I PKS KSs reveal the molecular details surrounding the evolution of substrate specificity and structural diversity, and their overall differences with canonical type I PKS KSs. Understanding the mechanism of substrate specificity will allow reprogramming of the KS active sites to generate polyketide analogues by PKS and polyketide biosynthetic pathway engineering. Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.