(HUMAN C6ORF130) IS AN O-ACYL-ADP-RIBOSE DEACYLASE

23 Pages·2011·5.14 MB·English

by  John Denu

JBC Papers in Press. Published on August 17, 2011 as Manuscript M111.276238
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.276238
ORPHAN MACRODOMAIN (HUMAN C6ORF130) IS AN O-ACYL-ADP-RIBOSE
DEACYLASE: SOLUTION STRUCTURE AND CATALYTIC PROPERTIES
Francis C. Peterson1§, Dawei Chen1¶, Betsy L. Lytle1§, Marianna N. Rossi#, Ivan Ahel#,
John M. Denu2¶, and Brian F. Volkman2§
From §Department of Biochemistry and Center for Eukaryotic Structural Genomics, Medical College of
Wisconsin, and ¶Department of Biomolecular Chemistry and Wisconsin Institute for Discovery,
University of Wisconsin-Madison, and #DNA Damage Response Group, Paterson Institute for Cancer
Research, University of Manchester, United Kingdom
Running Head: Structure and catalytic properties of O-acyl-ADP-ribose deacylase
1These authors contributed equally.
2Address correspondence to: John M Denu, 2178 Wisconsin Institute for Discovery, 330 N Orchard
Street, Madison, WI 53715. Fax: [phone]; E-mail: [email protected] or
Brian F Volkman, Department of Biochemistry and Center for Eukaryotic Structural Genomics, Medical
College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Fax: [phone]; Email:
[email protected]

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Post-translational modification of residues, Ser35 and Asp125. We propose a o
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proteins/histones by lysine acylation has catalytic mechanism for deacylation of O-acyl- lo
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profound effects on the physiological function ADP-ribose by C6orf130 and discuss the de
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of modified proteins. Deacylation by NAD+ biological implications in the context of fro
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dependent sirtuin reactions yields O-acyl-ADP- reversible protein acylation at lysine residues. h
ribose as a product, which has been implicated ttp
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as a signaling molecule in modulating cellular The macrodomain is an evolutionarily conserved w
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processes. Macrodomain-containing proteins protein fold found in isolation or embedded within .jb
are reported to bind NAD+-derived metabolites. larger polypeptides in the genomes of bacteria, c.o
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Here, we describe the structure and function of archaea, and eukaryotes (1). Named for their b/
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an orphan macrodomain protein, human presence in the C-terminus of the large variant g
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C6orf130. This unique 17-kDa protein is a core histone macroH2A (2), macrodomains adopt es
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stand-alone macrodomain protein that occupies a globular α/β-fold of ~140 residues that contain a n
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a distinct branch in the phylogenic tree. We deep binding cleft which serves as a ligand o
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demonstrate that C6orf130 catalyzes the binding site (3,4). Macrodomain containing m
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efficient deacylation of O-acetyl-ADP-ribose, O- proteins participate in a diverse array of cellular r 2
2
propionyl-ADP-ribose, and O-butyryl-ADP- functions (4,5), and recent reports have shown that , 2
0
ribose to produce ADPr and acetate, several macrodomains can bind NAD+ metabolites 18
propionate, and butyrate, respectively. Using including ADP-ribose (ADPr)3, poly(ADPr), and
NMR spectroscopy, we solved the structure of 2-O-acetyl-ADP-ribose (OAADPr) (6-9). Eleven
C6orf130 in the presence and absence of ADPr. macrodomain proteins encoded by 10 genes
The structures showed a canonical fold with a (7,10,11) have been identified in humans.
deep ligand (ADPr) binding cleft. Structural Structural studies of the ADPr-binding core of
comparisons of apo-C6orf130 and the ADPr- histone variant macroH2A1.1 in complex with
C6orf130 complex reveal fluctuations of the β - ADPr have shed light on macroH2a1.1-dependent
5
α loop that covers the bound ADPr, suggesting chromatin rearrangements upon PARP1 activation
4
that the β -α loop functions as a gate to (12). In addition, structural genomics research has
5 4
sequester substrate and offer flexibility to generated structures of two of the three
accommodate alternative substrates. The macrodomain-containing poly(ADPr) polymerases
ADPr-C6orf130 complex identified amino-acid (PARP14 and PARP15) in complex with ADPr
residues involved in substrate binding, and (PDB codes, 3Q71 and 3KH6, respectively;
suggested residues that function in catalysis. unpublished). No structural information is
Site-specific mutagenesis and steady-state available for the human macrodomain proteins
kinetic analyses revealed two critical catalytic GDAP2 or CHD1L (ALC1).
1

  Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Our research has focused on the function vaccines. A molecular understanding of the
of the novel metabolite, OAADPr. OAADPr is association of C6orf130 with chronic lymphocytic
produced in protein deacetylation reactions leukemia is not understood. In a recent proteomic
catalyzed by NAD+-dependent protein analysis, the putative C6orf130 ortholog from
deacetylases (13,14), which regulate gene Torpedo californica was identified in the electric
silencing, metabolic enzymes, and life span (15- organ (25). To understand the molecular function
18). OAADPr has been implicated as a signaling of this distinct member of the macrodomain
molecule, modulating cellular processes and family, we investigated the catalytic and structural
harnessing the regulation of metabolism and gene properties of human C6orf130.
activity (19-21). Recently we have shown that a Here we report the solution structures of
group of macrodomain proteins, i.e. MacroD-like C6orf130 in the apo and ADPr bound states using
proteins, including human MacroD1, human NMR spectroscopy as well as the biochemical
MacroD2, E. coli YmdB, and the sirtuin-linked characterization of its catalytic properties. In this
SAV0325 protein from S. aureus, function as study we show that C6orf130 is a new member of
OAADPr deacetylases, catalyzing the the OAADPr deacetylase family that efficiently
deacetylation of OAADPr to form ADP-ribose and catalyze the hydrolysis of OAADPr to produce
acetate (9). Mutagenesis and modeling of ADPr ADP-ribose and free acetate. We also show that
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into the putative active site of MacroD1 suggested C6orf130 can catalyze the deacylation of ADPr o
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important roles for Asn171, Asn174, Asp184, and derivatives with longer acyl chains, including O- lo
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His188. Unlike other human macrodomain propionyl-ADP-ribose (OPADPr) and O-butyryl- de
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proteins, MacroD1 and MacroD2 contain no other ADP-ribose (OBADPr), to produce ADPr and the fro
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known functional domains. The OAADPr corresponding acyl acids. To our knowledge, the h
deacetylation activity functionally links the C6orf130-ADPr NMR structure is the first of a ttp
MacroD-like proteins with the NAD+ dependent binary macrodomain complex that harbors O-acyl- ://ww
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protein deacetylases, connecting metabolic ADP-ribose deacylase activity, revealing key .jb
regulation with chromatin structure and gene residues for substrate binding and catalysis. c.o
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activity (5-7,22). Structural analyses of C6orf130 in the apo and b/
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C6orf130 is a unique stand-alone 17-kDa ADPr bound states indicate that the β5-α4 loop in gu
human macrodomain protein of unknown function. the core macrodomain fold functions as a lid to es
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The 152-amino acid protein is encoded by the sequester ADPr in the ligand-binding cleft. Based n
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open reading frame 130 of chromosome 6 (23). It on the structural, mutagenic, and catalytic studies, o
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shares the least amino acid sequence similarity to we propose a minimal catalytic mechanism for the m
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the other MacroD-like proteins, among which deacylation reaction of O-acyl-ADP-ribose by r 2
2
human MacroD1 and MacroD2 are closely related C6orf130. Comparisons with the MacroD-like , 2
0
and share more similarity to E. coli YmdB and the proteins indicate that the sequence variation within 18
sirtuin-linked SAV0325 protein from S. aureus. the macrodomain family permits a different set of
The diverged amino acid sequence of C6orf130 catalytic residues to perform O-acyl-ADPr
makes it a distinct branch on the phylogenetic tree hydrolysis.
(9). C6orf130 transcription and expression is
enriched in chronic lymphocytic leukemia cells EXPERIMENTAL PROCEDURES
and in B-cells of the chronic lymphocytic
leukemia patients when effective graft-versus- Materials—Tritium-labeled acetyl-CoA was
leukemia responses are achieved after donor purchased from Moravek Biochemicals (Brea,
lymphocyte infusion (24). C6orf130 can therefore CA). Synthetic peptide and acyl-peptides
be used as a biomarker for chronic lymphocytic corresponding to the 11 amino acids surrounding
leukemia and for monitoring the effectiveness of lysine-14 of histone H3, H N-KSTGGKAPRKQ-
2
adoptive immunotherapy for this disease. The high CONH (11-mer H3 peptide), H N-KSTGGK(-
2 2
level expression of C6orf130 in chronic acetyl)APRKQ-CONH , H N-KSTGGK(-
2 2
lymphocytic leukemia suggests the possibility of propionyl)APRKQ-CONH , H N-KSTGGK(-n-
2 2
using C6orf130 as a potential immunogen for butyryl)APRKQ-CONH were synthesized in the
2
developing chronic lymphocytic leukemia-specific University of Wisconsin-Madison Biotechnology
2
Center Peptide Synthesis Facility. Tritium-labeled mM Tris (pH 7.3) at 23 °C. Reactions were
acetyl H3 peptide, H N-KSTGGK(3H- initiated by addition of enzyme and terminated by
2
acetyl)APRKQ-CONH , was synthesized mixing an aliquot of the reaction mixtures with 1.5
2
enzymatically from the 11-mer H3 peptide and volumes of activated charcoal slurry at desired
purified as previously described (26). O-acetyl- time points. Inhibition was analyzed by measuring
ADP-ribose (OAADPr), O-propionyl-ADP-ribose the initial forward rate of 3H-acetate formation as
(OPADPr), O-butyryl-ADP-ribose (OBADPr), and described (9). The initial velocity data were fit
O-3H-acetyl-ADP-ribose (3H-OAADPr) were and displayed using Kaleidagraph (Synergy
synthesized enzymatically from ADPr and the Software, Reading, PA).
acyl-peptides using yeast deacetylase HST2 and C6orf130 expression and purification for
nicotinamidase from Salmonella enterica fused to structural studies—NMR samples of the C6orf130
maltose-binding protein (MBP–PncA) following gene product from H. sapiens were initially
procedures described previously (9,14). All other prepared in uniformly-13C, 15N labeled form
chemicals used were of the highest purity according to CESG wheat germ cell-free protocols
commercially available and were purchased from as described previously(29). Briefly, the protein
Sigma (St. Louis, MO), Aldrich (Milwaukee, WI), was expressed with an N-terminal His fusion tag
6
or Fisher Scientific (Pittsburgh, PA). in wheat germ extract supplemented with
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Protein expression and purification for enzymatic uniformly-13C, 15N labeled amino acids o
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assays—Expression and purification of yeast (Cambridge Isotope Labs) and purified by metal lo
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HST2 (14,27) and nicotinamidase from affinity chromatography followed by size- de
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Salmonella enterica fused to maltose-binding exclusion chromatography (SEC) as previously fro
m
protein (28) were performed as described described (30). Samples prepared using cell-free h
previously (9). Expression and purification of the protocols were used to solve the structure of apo- ttp
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His-tagged C6orf130 protein for enzymatic studies C6orf130. w
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was achieved by transforming E. coli BL21(DE3) Additional NMR samples for ADPr .jb
cells with the pDEST17 plasmid containing the titrations and structure determination of the ADPr– c.o
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C6orf130 gene insert and induction of mid-log C6orf130 complex were prepared recombinantly b/
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phase cells (A600 = 0.7) with 0.1 mM isopropyl-1- in E. coil strain SG13009[pRPEP4] (Qiagen) using gu
thio-β-D-galactopyranoside at 23 °C. Specific the pQE308HT vector as previously described es
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point mutations were created using the (31). Briefly, cells were grown at 37 °C in M9 n
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QuickChange II site-directed mutagenesis kit minimal broth containing 150 µg/ml of ampicillin ov
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(Stratagene). Mutated genes were sequenced at the and 50 µg/ml of kanamycin until reaching a cell m
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University of Wisconsin Biotechnology Center density of A600 = 0.7. Protein expression was then r 2
2
DNA Sequencing Facility to ensure that only the induced by the addition of IPTG to a final , 2
0
desired mutations were introduced. All protein concentration of 1 mM. Cells were grown for 18
purification was performed at 4°C or on ice as another 5 h, harvested by centrifugation and stored
described previously. The purified protein aliquots at -80 °C until processed further. Isotopically-
were re-concentrated, flash-frozen in liquid labeled proteins were prepared for NMR by
nitrogen, and stored at −80 °C. supplementing the M9 medium with 15N-
Enzyme Activity and Inhibition Assays—Enzyme ammonium chloride and/or 13C-glucose as the sole
activity assays for the deacylation of OAADPr, nitrogen and carbon sources, respectively. Cells
OPADPr, and OBADPr to ADPr and the harvested from a 1 L culture were lysed with a
corresponding acyl acids were performed by French pressure cell and purified by metal affinity
radioactive and HPLC methods using conditions chromatography using a previously published
as described (9). Inhibition of C6orf130 activity by protocol with one modification (32). In place of a
ADPr and the non-hydrolysable OAADPr analog second round of metal affinity chromatography,
N-acetyl-ADP-ribose (NAADPr) were assayed in the proteolytically cleaved His8 affinity tag was
reactions containing from 20 to 1,600 µM 3H- separated from C6orf130 using a Sephacryl-100
OAADPr, 0.5 µM C6orf130 enzyme, and ADPr SEC column equilibrated with 50 mM Bis-Tris,
concentrations ranging from 0 to 400 µM or pH 6.5 and 200 mM sodium chloride.
NAADPr concentrations from 0 to 500 µM in 50
3
NMR spectroscopy—NMR samples were prepared CYANA structures calculated, the 20 conformers
in buffer containing 50 mM Bis-Tris at pH 6.5, with the lowest target function were subjected to a
200 mM sodium chloride, 2 mM dithiothreitol, molecular dynamics protocol in explicit solvent
and 5-10% 2H O. All NMR data were acquired at (40) using XPLOR-NIH (41). The appropriate
2
25°C on a Bruker 600 MHz spectrometer equipped topology and parameter files for ADPr were
with a triple-resonance CryoProbeTM and generated in a semi-automated manner using the
processed with NMRPipe software (33). ADPr PRODRG server
(Sigma) was incrementally titrated into a sample (http://davapc1.bioch.dundee.ac.uk/prodrg/) and
containing 250 µM 15N-labeled C6orf130 and manually edited to add the omitted protons (38).
monitored by 2D 15N, 1H-HSQC to confirm The apo-C60rf130 and ADPr–C6orf130 structures
binding. Backbone 1H, 15N, and 13C chemical shift were deposited in the Protein Data Bank, PDB IDs
assignments for apo-C6orf130 were obtained 2LGR and 2L8R, respectively, and the Biological
automatically as previously described using a 550 Magnetic Resonance Bank (BMRB), BMRB IDs
µM sample of 15N, 13C labeled apo-C6orf130 15593 and 17421, respectively.
(34,35). Sidechain assignments were completed
manually from 3D HBHACONH, HCCONH, RESULTS
HCCH total correlation spectroscopy, and
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13C(aromatic)-edited NOESY-HSQC spectra. C6orf130 deacetylase activity—Eleven o
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Backbone and sidechain assignments were macrodomain proteins encoded by 10 genes lo
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transferred to the ADPr–C6orf130 complex by (7,10,11) have been identified in humans. Two of de
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inspection and confirmed with 3D HNCACB and the human macrodomain proteins, MacroD1 and fro
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HCCH total correlation spectroscopy acquired MacroD2, are structurally and functionally h
using a 800 µM sample of 15N,13C labeled ADPr– homologous to each other and are closely related ttp
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C6orf130. Chemical shift assignment were ~91% to bacterial YmdB proteins and to the sirtuin- w
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and ~93% complete for the apo and holo proteins, linked protein SAV0325 from pathogenic .jb
respectively. Heteronuclear NOEs values were bacterium S. aureus (Supplemental TableS1). As a c.o
measured from an interleaved pair of 2D 15N-1H member of the macrodomain superfamily, brg/
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sensitivity enhanced correlation spectra recorded C6orf130 shares minimal homology with the g
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with and without a 5s proton saturation period. MacroD-like proteins including human MacroD1 es
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The combined chemical shift difference for each and MacroD2 (Figure 1a, Supplemental TableS1). n
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residue was calculated as [((ΔδN/5)2 + (ΔδH)2)/2]1/2, C6orf130 transcription and expression are ove
where ΔδN and ΔδH are the 15N and 1HN chemical enriched in chronic lymphocytic leukemia cells, mbe
shift differences (ppm), respectively, between the suggesting this protein might be a promising r 2
2
two conditions. serological marker for this disease (24). To , 2
0
Structure calculations of apo-C6orf130 and explore the molecular and biological functions of 18
ADPr–C6orf130—Distance constraints were this unique macrodomain protein, we examined
obtained from 3D 15N-edited NOESY-HSQC and whether C6orf130 was capable of hydrolyzing
13C-edited NOESY-HSQC (τ = 80 ms). OAADPr and other NAD+ metabolites derived
mix
Intermolecular distance constraints between from the sirtuin reaction.
C6orf130 and ADPr were obtained from a 3D F1- By incubating radiolabeled O-3H-acetyl-
13C-filtered/F3-13C-edited NOESY-HSQC ADP-ribose with purified C6orf130 and analyzing
spectrum (τ = 120 ms) (36). Backbone φ and ψ the incubation mixture using radioactive charcoal-
mix
dihedral angle constraints were generated from binding and HPLC (9), we found that C6orf130
secondary shifts of the 1H, 13Cα, 13Cβ, 13C’ and 15N carries a robust catalytic activity towards
nuclei using the program TALOS (37). A CYANA deacetylation of OAADPr, forming ADPr and
library file was generated for the ADPr ligand as acetate. Figure 1b shows that the initial rates of
we have previously described for small molecules OAADPr deacetylation catalyzed by C6orf130
(38). Structure calculations were performed using depend on the concentration of OAADPr and
the torsion angle dynamics program CYANA (39) exhibit saturation kinetics. An apparent Km of 182
followed by iterative rounds of manual refinement ± 17 µM and Vmax of 0.31 ± 0.03 s−1 at 23°C were
to eliminate constraint violations. Of the 100 determined (Table 1). To investigate the substrate
4
specificity and whether C6orf130 also possesses of active site residues. Inhibition by ADPr also
catalytic activity towards other sirtuin-derived suggests that ADPr could act as an in vivo
NAD+ metabolites, we synthesized longer acyl inhibitor of macrodomain-dependent OAADPr
derivatives of ADPr and used them as alternative deacetylation and provides feedback regulation of
substrates. Catalytic assays using the alternative cellular OAADPr concentration. The relative
substrates show that C6orf130 efficiently larger inhibition constant of NAADPr suggests
catalyzed the deacylation of OPADPr and that the rigidity of the amide bond of NAADPr is
OBADPr to form ADPr and propionate and less favored at the substrate-binding site.
butyrate, respectively. The deacylation of Structure of the apo-C6orf130 macrodomain—To
OPADPr and OBADPr by C6orf130 followed investigate whether C6orf130 retains the canonical
similar saturation kinetics. Increasing the acyl macrodomain fold, we determined its solution
chains of O-acyl-ADPr only slightly increased the structure using standard NMR methods (35). The
K and decreased catalytic turnover k . The final ensemble of 20 conformers is shown in
m cat
steady-state kinetic parameters for deacylation of Figure 2a and the structural statistics are presented
OPADPr, OBADPr, and OAADPr are summarized in Table 2. Like other macrodomains, C6orf130
in Table 1. The data indicate that C6orf130 has a exhibits the canonical three-layered α -β-α
slight preference for ADPr derivatives with shorter sandwich with a central six-stranded β -sheet
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acyl chains. containing a mixture of anti-parallel (β -β -β ) and o
3 4 2 w
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Inhibition of C6orf130 by ADPr and an OAADPr parallel (β2-β5-β6-β1) strands and a deep ligand loa
Analog—Previous x-ray crystallography and binding cleft (Figure 2b). Structural alignment of de
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biochemical studies revealed that human C6orf130 with the macroD1 and macroH2A1.1 fro
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macroH2A1.1 functions as a binding module for macrodomains reveals that C6orf130 contains only h
ADPr, OAADPr and other analogs (6,22). Human the core domain and lacks the extended N- and C- ttp
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MacroD1 and MacroD2 bind ADPr with high terminal structural elements commonly found in w
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affinity (7) and ADPr serves as a competitive other macrodomains (Figure 2c) (9,42). .jb
inhibitor for OAADPr deacetylation catalyzed by A Dali search (43) using the apo C6orf130 c.o
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MacroD1 (9). Here, we examined the ability of structure revealed the presence of numerous b/
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ADPr and a non-hydrolyzable substrate analog N- structural homologs. The top scoring homologs are g
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acetyl-ADP-ribose (NAADPr) to function as a hypothetical protein BT1257 from Bacteroides es
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inhibitors for the deacetylation of OAADPr by thetaiotaomicron (PDB codes, 2FG1 and 2AFC; n
N
C6orf130. Deacetylation reactions of OAADPr Z-scores of 17.0 for both; RMSDs of 2.3 Å and o
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were initiated by addition of C6orf130 in the 2.1 Å for 140 and 137 superimposed Cα atoms, m
b
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presence of 0-400 µM of ADPr or 0-500 µM of respectively); a hypothetical protein TTHA0132 r 2
2
NAADPr and the initial rates of acetate formation from Thermus thermophilus (PDB code, 2DX6; Z- , 2
0
were measured under steady-state conditions. As score of 14.9; RMSD of 2.5 Å for 134 18
shown in Figure 1c and d, increasing the superimposed Cα atoms); the nsP3 macro domain
concentration of ADPr or NAADPr resulted in an of Chikungunya virus in complex with ADP-
increase of the apparent Michaelis constant, K , ribose (PDB code, 3GPO; Z-score of 13.2; RMSD
m
while the maximum catalytic rates were minimally of 2.6 Å for 131 superimposed Cα atoms); and
affected. These data are consistent with reversible core histone macroH2A1.1 (PDB code, 1YD9; Z-
competitive inhibition of C6orf130 by ADPr and score of 12.6; RMSD of 2.7 Å for 138
NAADPr. Inhibition constants (K) of 119.3 ± 4.5 superimposed Cα atoms).
i
µM and 223.5 ± 11.3 µM were determined at 23 Low backbone RMSD and high 15N-1H
°C for ADPr and NAADPr, respectively. These heteronuclear NOE values (Figure 2d) over the
results suggest that C6orf130 contains a specific length of C6orf130 suggest the protein is well
binding site for ADPr and NAADPr, which is ordered with two exceptions. The sharp increase in
identical to the active site for OAADPr RMSD values and concurrent decrease in 15N-1H
deacetylation. Inhibition of OAADPr heteronuclear NOE values for the first 14 residues
deacetylation by ADPr indicates that structural indicates that the N-terminus is unstructured and
information from a C6orf130-ADPr binary dynamically disordered. This conclusion is
complex can be used to investigate the functions supported by the lack of long range NOEs for
5
these residues in NOESY spectra (data not binding and catalysis, we solved the NMR solution
shown). A similar increase in RMSD values and structure of the ADPr–C6orf130 complex. The
lack of long range NOEs is observed for residues final ensemble of 20 conformers is shown in
119-129 in the loop connecting β-strand 5 to α- Figure 4a and the structural statistics are presented
helix 4 (β -α loop), but in contrast to the N- in Table 2. ADPr binds in the deep cleft of
5 4
terminus 15N-1H heteronuclear NOE values remain C6orf130 and is covered by the β -α loop (Figure
5 4
uniformly high. Taken together, the disparity 4b), consistent with the chemical shift perturbation
between RMSD and 15N-1H heteronuclear NOE data (Figure 3b). RMSD and 15N-1H heteronuclear
values and the line broadening observed for β -α NOE values for the ADPr–C6orf130 complex
5 4
15N-1H HSQC signals indicate that this loop (Figure 4c) are largely unchanged when compared
undergoes conformational exchange on an with those for apo-C6orf130 (Figure 2d) except in
intermediate (µsec-msec) time scale. the β -α loop. In the complex, RMSD values for
5 4
NMR analysis of ADPr binding to C6orf130—Our residues 119-129 in the β -α loop decreased to
5 4
enzymatic studies demonstrate that C6orf130 values consistent with all other well-ordered
catalyzes the deacylation of O-acylated-ADPr to residues. The low RMSD values and high 15N-1H
yield ADPr and the corresponding organic acid. heteronuclear NOE values indicate that the β -α
5 4
Inhibition studies indicate that ADPr serves as a loop fluctuations present in the apo state were
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competitive inhibitor for OAADPr binding (Fig dampened upon ADPr binding. The appearance of o
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1c). To determine whether the conserved binding long range NOEs between the methyl groups of lo
a
cleft functions as the substrate recognition site, we L124 and the side chains of A42 and G43 (Figure de
d
incrementally titrated 15N-labeled C6orf130 with 4d) reinforces this conclusion and confirms that fro
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AInDspPerc tiaonnd ofm tohne itroerseudlt inthge 1 5Nch-1aHng HesS QbCy sNpeMctRra. wthiet hβin5 -tαh4e Clo6oopr f1e3n0cl aocsteisv eA sDiteP. r or other ligands http
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identified approximately 20 resonances that Mutational analysis of the C6orf130 active site— w
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respond in a dose-dependent manner to ADPr The structure of the C6orf130-ADPr binary .jb
(Figure 3a). Movement of only a subset of peaks complex revealed amino acid residues interacting c.o
rg
indicated that the interaction with ADPr is specific with the bound ADPr (Figure 5a) and suggested b/
y
and utilizes a defined binding site. The ADPr- residues important for catalysis. The side chain g
u
induced chemical shift perturbations localized to carboxyl group of Asp20 interacts with the es
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the loop connecting β-strands 1 and 2, β-strand 2, adenine primary amine of the bound ADPr. Leu21 n
N
the N-terminal end of α-helix 2 and the β5-α4 loop and Phe22 on the β1-β2 loop, Ile44, Leu47 on the ove
(Figure 3b). Mapping these perturbations to the β2-α1 loop, Pro118, Tyr150, and the C-terminal mbe
three-dimensional structure of apo-C6orf130 Leu152 form a hydrophobic pocket in which the r 2
2
confirmed that the deep cleft conserved in the core adenosine moiety is embedded. The electrostatic , 2
0
macrodomain fold serves as the ADPr binding and hydrophobic interactions between the 18
pocket and residues that exhibited the largest adenosine moiety and the protein binding pocket
perturbations (> 0.6) lined the floor of the cleft seem to contribute the majority of the binding
(Figure 3c and d). The majority of residues in the energy, as indicated by the higher coordinate
β5-α4 loop exhibit perturbations above the precision of the adenosine side compared to the
threshold value (≥ 0.25). The sensitivity of the β5- distal ribose side of the bound ADPr (Figure 4a).
α4 loop to ADPr binding, its proximity to the The GXG sequence in the β5-α4 loop is positioned
ligand binding groove, and the conformational over the pyrophosphate group and is a common
fluctuation on the intermediate time scale suggests phosphate binding element found in numerous
that the β5-α4 loop may serve as a gate that limits nucleotide binding proteins (44). The side chains
substrate binding or product release from the of Ser35, Thr83, and Asp125 are located in
ligand binding cleft. Depending on its vicinity of the distal ribose and potentially interact
conformation in the substrate-bound state, specific with the C2- and C3- hydroxyl groups. His32 and
β5-α4 residues may also participate in O-acyl- Cys33 are also located nearby and their main chain
ADPr hydrolysis. amides form part of the substrate-binding pocket.
Structure of ADPr–C6orf130 complex—To further To verify that the ADPr binding pocket
investigate the role of the β5-α4 loop in substrate constitutes the substrate-binding site and to
6
identify important residues for catalysis, we state kinetic parameters of the T83A and S35A
performed site-directed mutagenesis on potentially mutants were determined (Figure 5b) and were
important residues for substrate binding and compared with those of the wild-type enzyme
catalysis. Gly123 was substituted with Glu while (Table 3). Taken together the data indicate that
His32, Cys33, Ser35, Thr83, and Asp125 were Ser35 and Asp125 function as critical catalytic
substituted with Ala. The purified mutant residues. A proposed catalytic mechanism is
C6orf130 proteins were assayed for OAADPr discussed below.
deacetylation activity. Mutants retaining
deacetylation activity were further analyzed under DISCUSSION
steady-state conditions. During the purification of
these C6orf130 variants, no abnormal elution Comparison of C6orf130 with other macrodomain
behaviors were observed in the Sephadex G75 gel proteins—C6orf130 occupies a distinct branch in
filtration chromatography, suggesting that amino the phylogenic tree of macrodomain proteins (9).
acid substitution at these positions did not cause Despite its low sequence homology to MacroD-
global structural perturbations. Substituting Cys33 like proteins (Figure 1a, supplemental Table S1),
with Ala affected neither substrate binding nor C6orf130 exhibits a similar canonical core fold as
catalytic efficiency. Substituting His32, Gly123, other macrodomain proteins, consisting of three-
D
and Asp125 with Ala resulted in enzymes whose layered α-β-α sandwich with a central six-stranded o
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measured activities were within the rates observed β-sheet containing a mixture of anti-parallel (β3- loa
for background hydrolysis. From the C6orf130- β -β ) and parallel (β -β -β -β ) strands and a de
4 2 2 5 6 1 d
ADPr structure, the main chain NH of Gly123 ligand binding cleft (Figure 2b). Structural fro
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hydroxyls of adenosine. Introducing a large side domain and lacks the extended N- and C-terminal w
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chain with a negative charge in the G123E mutant structural elements found in MacroD1 and .jb
presumably disrupts the substrate binding and MacroH2A1.1 (Figure 2c) (9,42). In the apo c.o
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renders the variant inactive. The imidazole ring of structure of human MacroD1, an ADPr molecule b/
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His32 is buried in a hydrophobic pocket that is was modeled in the ligand binding cleft (9) with g
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formed by the side chains of Phe48 and Try80. the diphosphate ribose moiety and distal 2- and 3- es
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While one edge of the imidazole ring comes hydroxyl groups positioned at the surface of the n
N
within ~3 Å of the pyrophosphate, the main role of protein in a highly solvent-accessible location. In o
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His32 may be to maintain the integrity of the contrast, the C6orf130-ADPr costructure solved in m
b
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substrate binding pocket. The carboxyl of Asp125 this study reveals that ADPr binds deep within the r 2
2
is within H-bonding distance to the C2- and C3- macrodomain cleft with the diphosphate ribose , 2
0
hydroxyl groups of the distal ribose (i.e. ADP- portion completely buried under the β -α , β -α , 18
2 1 4 3
ribose-C2(OH) and C3(OH)). The D125A mutant and the β -α loops. The canonical core fold and
5 4
showed no detectable deacetylation activity after similar deacetylation activity towards OAADPr
subtracting background activity obtained with suggest a convergent evolution of the catalytic
rates in the absence of enzyme. No OAADPr function between C6orf130 and the MacroD-like
deacetylation activity was detected using enzyme proteins.
concentrations as high as 5 µM with prolonged Catalytic mechanism of C6orf130—ADPr acts as a
incubation. The S35A point mutation resulted in ~ competitive inhibitor of C6orf130 deacylation,
20 fold decrease in k without significantly suggesting that the substrate O-acyl-ADPr binds in
cat
affecting K (k = 0.023 ± 0.002 s-1, K = 177.7 ± the same fashion as ADPr. The mobile β -α loop
m cat m 5 4
12.6 (µM), and V/K = (1.29 ± 0.15) × 102 M-1s-1). appears to function as a gate that combines with
Thus, substituting Ser35 with Ala lowers the first- the β -α and β -α loops to sequester substrate
2 1 4 3
order rate constant for the chemical conversion of from the solvent environment. On these loops,
the enzyme-substrate complex but does not affect Ser35 (β -α ), Thr83 (β -α ), and Asp125 (β -α )
2 1 4 3 5 4
substrate binding as assessed by an unaffected K are located close to the C2- and C3- hydroxyls of
m
value. The T83A substitution yielded an ~ 3 fold the distal ribose, serving potentially important
decrease in k without affecting the K . Steady-
cat m
7
roles in catalysis. The present mutagenic studies hydrolytic efficiency. The observation that the
revealed that the T83A mutant retains nearly all of catalytic residues between C6orf130 and MacroD-
its deacetylation activity relative to wild type like proteins are not conserved indicates sequence
enzyme; therefore, the possibility of Thr83 variation within the macrodomain family permits a
functioning as the key catalytic residue can be different set of catalytic residues to perform O-
ruled out. Substituting Ser35 with Ala severely acyl-ADPr hydrolysis.
impaired the catalytic capability with a ~20-fold Macrodomains as general deacylases of O-acyl-
decrease in k . However, the K of the S35A ADP-ribose—Protein lysine acetylation can have
cat m
mutant is minimally affected (Table 3). The most profound effects on the physiological function of
dramatic substitution was the Asp125 to Ala modified proteins. Propionylation, butyrylation,
mutant, which resulted in a C6orf130 variant with and succinylation of lysine represent newly
no detectable deacetylation activity. Thus, Ser35 discovered protein post-translational modifications
and Asp125 are candidate catalytic residues. (45-47). Although it is unclear which enzymes are
Steady state kinetic analyses of these mutant responsible for the addition of these groups,
enzymes and the structural information from both human acetyltransferases p300 and CBP catalyzed
apo-C6orf130 and C6orf130-ADPr complex lead in vitro lysine propionylation and butyrylation of
us to propose a minimal mechanism for the histones (45,46). Berndsen et al. showed that the
D
C6orf130-catalyzed deacylation reaction (Figure yeast histone acetyltransferase Esa1 could readily o
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6c), bearing in mind that S35A retains ~5% kcat perform propionyl transfer from propionyl-CoA to loa
activity of the wild type enzyme and there are no histones (48). In Salmonella enterica, de
d
other functional groups in the vicinity of the C2 propionylation of propionyl-CoA synthetase was fro
m
and C3 hydroxyls of the distal ribose (ADP- catalyzed by prokaryotic Gcn-5-related N- h
ribose-C2(OH) and C3(OH)). In this mechanism acetyltransferases (28). Thus, known ttp
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the hydroxyl of Ser35 is H-bonded to the ester acetyltransferases might catalyze these reactions in w
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carbonyl oxygen to polarize the carbonyl bond. vivo, utilizing cellular propionyl-CoA, butyryl- .jb
With Asp125 acting as a general base, a water CoA, and succinyl-CoA as the cosubstrates. The c.o
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molecule is activated and functions as a propionyl-lysine and butyryl-lysine in the b/
y
nucleophile to attack the carbonyl carbon and form modified proteins can be deacylated in vitro by g
u
an oxyanion tetrahedral intermediate. Protonation NAD+ dependent reactions catalyzed by sirtuins, es
t o
of the scissile ester oxygen atom and producing O-acyl-ADPr as the coproduct n
N
rearrangement result in the breakdown of the (19,28,46,49). These observations suggest that o
v
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tetrahedral intermediate to form ADPr and the propionyl-, butyryl-, and succinyl-lysine proteins m
b
e
corresponding acyl acids (Figure 6c). might be natural substrates of sirtuins in vivo, r 2
2
Previous kinetic analysis of MacroD1 although desuccinylation has yet to be reported. , 2
0
mutants suggested that Asn171, Asn174, Asp184, Acetyl-CoA, propionyl-CoA, butyryl-CoA and 18
and His188 are important for catalysis. Modeling succinyl-CoA are important intermediates in
of ADPr into the solved MacroD1 structure cellular energy metabolism, fatty acids β-
located these residues near the C2 and C3 oxidation, and the degradation of leucine, lysine,
hydroxyls of the distal ribose (9). Asn171 and isoleucine, valine, and methionine. The reversible
Asn174 are widely conserved among many acylation of proteins and histones by acyl-CoA
different macrodomains but not in C6orf130. dependent acyltransferases and the NAD+
Among MacroD-like proteins, Asp184 and His188 dependent protein deacylases connect energy
are conserved. In MacroD1, Asn174 was proposed metabolism with the functional (de)acylation of
to help position the ribose and polarize the ester targeted proteins, as well as the production of a
bond for hydrolysis. Asn171 and His188 interact diverse set of O-acyl-ADPr metabolites. O-acetyl-
with Asp184 and might facilitate deprotonation, ADP-ribose has been implicated in metabolic
allowing Asp184 to function as a general base by regulation, gene expression, cellular redox control
activating a water molecule for nucleophilic attack and cation channel activity (5-7,19-22,50,51).
on the carbonyl carbon. In contrast to Ser35 and Thus, the O-acyl-ADPr deacylation activity of
Asp125 from C6orf130, the four catalytic residues C6orf130 and the recently characterized MacroD-
in MacroD1 individually contribute little to like proteins place these enzymes in a pivotal
8
position to regulate the functions of various O- catalytic residues. ADPr binding dampens the
acyl-ADPr metabolites. The ability of C6orf130 fluctuations of the β -α loop closing the “lid”
5 4
to deacylate O-acyl-ADPr with varying acyl sequestering ADPr or other ligands in the
chains suggests that C6orf130 might function as C6orf130 active site (Figure 6a). “Lid” closure
an in vivo modulator of cellular processes forms a pliable pore through which longer acyl
regulated by the reversible acylation of proteins chains, such as the modeled butyryl moiety can be
and histones (catalyzed by the acyl-CoA accommodated in the C6orf130 (Figure 6b).
dependent acyl transferases and the NAD+ Together, our structural studies of apo-C6orf130,
dependent deacylases). the C6orf130-ADPr complex, and modeling of
The ability of C6orf130 to accept acyl- longer acyl-chain ADPr esters are consistent with
ADPr substrates with longer acyl chains can be the observed catalytic properties using OAADPr,
explained from the structures solved in this study. OPADPr, and OBADPr as substrates.
In the C6orf130-ADPr binary complex, the
substrate is buried by flexible loops carrying the

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