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Review
. 2015 Jun 10;115(11):5413-61.
doi: 10.1021/acs.chemrev.5b00003. Epub 2015 May 13.

Chemical biology of protein arginine modifications in epigenetic regulation

Jakob Fuhrmann  1 , Kathleen W Clancy, Paul R Thompson
Affiliations
Review

Chemical biology of protein arginine modifications in epigenetic regulation

Jakob Fuhrmann et al. Chem Rev. .
No abstract available

PubMed Disclaimer

Figures

Figure 1
Figure 1
N-terminal tails of histone proteins are the preferred targets of histone-modifying enzymes. The major modifications of histone arginine residues are citrullination and methylation. Abbreviations: Cit, citrulline; MMA, monomethylarginine; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine.
Figure 2
Figure 2
Bidendate interactions of the arginine guanidinium group exemplified by (i) the carboxyl group of aspartate (trypsin–peptide complex, PDB code 1OX1) (left), (ii) the phosphoryl group of phosphotyrosine (SH2–pTyr, PDB code 4F5B) (middle), and (iii) atoms O6 and N7 of guanine (p53–DNA, PDB code 3TS8) (right).
Figure 3
Figure 3
PAD enzymes catalyze the hydrolytic conversion of peptidyl arginine residues into citrulline.
Figure 4
Figure 4
Electrostatic surface potential and hydrogen bond donor/acceptor sites of the side chains of arginine and citrulline. Hydrogen bond donor sites are highlighted in red, whereas hydrogen bond acceptor sites are depicted in blue. Cα denotes the α-carbon. Charge potentials were rendered by using SPARTAN (Wavefunction Inc.), with negative electrostatic charges shown in red, positive charges in blue, and neutral charges in green.
Figure 5
Figure 5
Sequence alignment of human PAD family members. Catalytic residues are highlighted with red asterisks below the alignment. The sequence alignment was generated using Clustal Omega and visualized using Espript 3.0. The consensus sequence is abbreviated as follows: uppercase letters indicate identical residues, lowercase letters indicate consensus level >0.5, “!” represents any conserved residue of isoleucine (I) or valine (V), “$” represents any conserved residue of leucine (L) or methionine (M), “%” represents any conserved residue of phenylalanine (F) or tyrosine (Y), and “#” represents any conserved residue of asparagine (N), aspartate (D), glutamine (Q), or glutamate (E). The relative accessibility of each residue is depicted below the consensus motif: blue indicates accessible residues, cyan marks intermediately accessible residues, white stands for buried residues, and red indicates that the accessibility is not predicted.
Figure 6
Figure 6
Surface representation of the dimeric PAD4 C645A mutant bound to the substrate BAA (PDB code 1WDA). (A) PAD4 exists as a head-to-tail dimeric protein that comprises three domains as indicated for one protomer. (B) The catalytic sites of both protomers are located on the same dimer face and are separated by ∼65 Å. Abbreviation: Ig subdomain, immunoglobulin-like subdomain.
Figure 7
Figure 7
Domain organization and calcium-binding sites of the PAD4 C645A protomer bound to the substrate BAA (PDB code 1WDA). The structural elements are color coded according to Figure 6. The insets on the right depict two orientations of the PAD4 active site bound to BAA, highlighting critical residues for substrate binding and catalysis. Polar contacts of <3.5 Å are represented as dashed lines.
Figure 8
Figure 8
The image on top represents the structure of PAD4 without Ca2+ (blue) (PDB code 1WD8) superimposed onto the structure of PAD4 with Ca2+ (orange) (PDB code 1WD9). The image on the bottom depicts the structure of apoPAD2 without Ca2+ (gray) (PDB code 4N20) overlaid onto the structure of holoPAD2 F221/222A mutant with Ca2+ (purple) (PDB code 4N2C). The movements of active site residues (bold) are highlighted as dashed lines. Ca2+ ions are illustrated as green spheres, whereas active site residues are shown as sticks.
Figure 9
Figure 9
Top view of the PAD4 C645A mutant bound to BAA colored according to its electrostatic surface potential, highlighting two connected cavities that form a continuous tunnel (orange rod) of ∼21 Å (PDB code 1WDA). The lower image illustrates the side view of the active site cavity (front door), occupied by BAA, and the back door tunnel, presumably involved in incoming water channeling and ammonia (product) extrusion.
Figure 10
Figure 10
PAD4 (orange) bound to histone H3 peptide (gray) (PDB code 2DEW). Waters are depicted as red spheres. Adapted with permission from ref (31). Copyright (2006) National Academy of Sciences, U.S.A.
Figure 11
Figure 11
Proposed catalytic mechanism for PAD enzymes.
Figure 12
Figure 12
Comparison of PAD4 and DDAH active site pockets. (A) Active site of PAD4 with bound BAA (left side) (PDB code 1WDA), bovine DDAH with bound citrulline abbreviated as Cit (middle panel) (PDB code 2C6Z), and human DDAH with bound N5-(1-iminopentyl)-l-ornithine abbreviated as LN6 inhibitor (right side) (PDB code 3P8P). All protein structures are colored according to their electrostatic surface potential. (B) Bidendate recognition of the substrate arginine guanidinium group (gray) by the carboxyl groups of PAD4 (orange, PDB code 1WDA) D473 and D350, left panel. Methylation of the arginine guanidinium group would disfavor and preclude tight interactions with the aspartate residues, right panel.
Figure 13
Figure 13
(A) Active site of argininosuccinate synthetase with bound citrulline, aspartate, and ATP (PDB 1J1Z) and proposed mechanism. (B) Active site of the argininosuccinate lyase homologue δ crystalline T161D mutant with bound argininosuccinate highlighted in gray (PDB 1TJW) and proposed mechanism. Abbreviations: Cit, citrulline; Asp, aspartate; ATP, adenosine triphosphate; PPi, pyrophosphate; AMP, adenosine monophosphate, AS, argininosuccinate; H–B, general acid.
Figure 14
Figure 14
Reversible PAD inhibitors. The presence of a guanidine group is highlighted in blue.
Figure 15
Figure 15
(A) Reversible, mixed-type PAD4 inhibitors. Kis is the dissociation constant for the enzyme–inhibitor complex. (B) Crystal structure of PAD4 bound to inhibitor 9, GSK199 (PDB code 4X8G). GSK199 (gray) directly interacts with active site residues H471 and D473, and is further stabilized by binding to F634 and N588. Hydrogen bonds of <3.5 Å are represented as dashed black lines. (C) The image on the left side depicts the structure of PAD4 (orange) bound to inhibitor GSK199 (gray, PDB code 4X8G) superimposed onto the structure of PAD4 (green) bound to BAA substrate (green stick model, PDB code 1WDA). Residues 633–640 (red, denoted by α) of PAD4 bound to BAA adopt an α-helical conformation, while residues 633–645 (yellow, denoted by β) of PAD4 bound to GSK199 form an antiparallel β-sheet. The image on the right side compares the binding sites of BAA (green) and GSK199 (gray) mapped onto the structure of PAD4 (PDB code 1WDA), colored according to its electrostatic surface potential.
Figure 16
Figure 16
(A) Covalent, irreversible inhibitors of the PADs. The presence of an amidine group is highlighted in blue. The potency toward the individual PAD isozymes is represented below the compounds. (B) Potential mechanisms of PAD inactivation by chloroacetamidine-based inhibitors.
Figure 17
Figure 17
Crystal structures of PAD4 with inhibitors bound. (A) Schematic overview of PAD4 (blue) bound to a peptidyl substrate (gray) (left panel). Structural alignment of inhibitors bound to the active site of PAD4. The histone H3 peptide (sequence TARKS) bound to PAD4 (gray) (PDB code 2DEW) is included to compare the substrate-binding site. The Cl-amidine (magenta) (PDB code 2DW5), o-Cl-amidine (yellow) (PDB code 3B1T), and TDFA (red) (PDB code 4DKT) structures are aligned accordingly, including the depiction of critical PAD4-interacting residues. (B) Close-up view of PAD4 with bound inhibitors, highlighting critical inhibitor backbone interactions with PAD4 residues shown as dashed lines. Interactions between the amidine group as well as the α-amine of the inhibitor and PAD4 are omitted for clarity.
Figure 18
Figure 18
ABPP probes for PADs. (A) Structures of PAD-selective probes. The amidine group is highlighted in blue, whereas the reporter tags (rhodamine or biotin) are marked in red. (B) Schematic overview of fluorescence polarization assay using the PAD4-specific RFA probe.
Figure 19
Figure 19
Citrullination sites in histone proteins. Color code: green, gene activation; red, gene repression; yellow, gene activation or repression, or unknown.
Figure 20
Figure 20
NET formation in neutrophils.
Figure 21
Figure 21
Schematic depiction of the human PRMT family. The SAM-binding methyltransferase region is highlighted in olive green. All family members contain the methyltransferase signature motifs I, post-I, II, and III and the conserved THW loop, labeled as red bars, respectively. Sequence motifs with low or no sequence similarity are depicted in light red. Abbreviations: SH3, SH3 domain; Zn, zinc finger motif; TPR, tetratricopeptide repeat.
Figure 22
Figure 22
PRMTs are SAM-dependent enzymes that catalyze the transfer of methyl groups onto peptidyl arginine residues. There are three types of PRMTs that are classified according to the site of modification. Type I enzymes generate asymmetric dimethylations, type II enzymes form symmetric dimethylations, and the type III enzyme PRMT7 only catalyzes monomethylation reactions.
Figure 23
Figure 23
(A) Electrostatic surface potential and hydrogen-bonding donor sites of the side chain of arginine, and the methylated arginine side chains of MMA, ADMA, and SDMA. Cα denotes the α-carbon. Charge potentials were rendered by using SPARTAN (Wavefunction Inc.), with negative electrostatic charges shown in red, positive charges in blue, and neutral charges in green. (B) Distinct stereoisomers for MMA and SDMA. Methyl groups are highlighted in yellow, whereas hydrogen-bonding donor sites are marked in red. Stereoisomers emerging from rotation around the central Cζ–Nε bond are omitted for simplicity.
Figure 24
Figure 24
Sequence alignment of the SAM-binding methyltransferase region of human PRMT family members. The product specificity-determining residue is highlighted with a blue asterisk below the alignment. Catalytic residues located on the double E-loop are highlighted with red asterisks below the alignment. The sequence alignment was generated using Clustal Omega and visualized using Espript 3.0. The relative accessibility of each residue is depicted below the consensus motif: blue indicates accessible residues, cyan marks intermediately accessible residues, white stands for buried residues, and red indicates that the accessibility is not predicted.
Figure 25
Figure 25
(A) PRMT1 exists as a head-to-tail dimeric protein that comprises four characteristic functional regions, as indicated (PDB code 1OR8). (B) Surface representation of dimeric PRMT1 colored according to its electrostatic surface potential. The catalytic sites of both protomers are located on the same dimer side and are facing each other, separated by ∼30 Å.
Figure 26
Figure 26
Homodimeric PRMT1 contains two active sites and a central hole (PDB code 1OR8). Monomeric PRMT7 from Mus musculus harbors only one active site and does not possess a central hole (PDB code 4C4A). Homodimeric PRMT7 from T. brucei contains two active sites and does not possess a central hole (PDB code 4M37).
Figure 27
Figure 27
Active site architecture of PRMT1 bound to arginine (PDB code 1OR8). The structural elements are color coded according to Figure 25. The image on the right depicts details of the PRMT1 active site, highlighting critical residues for substrate binding and catalysis. Polar contacts of <3.5 Å are represented as dashed lines.
Figure 28
Figure 28
PRMT5·MEP50 (pink) complex bound to histone H4 peptide (gray) (PDB code 4GQB). Polar contacts of <3.5 Å are represented as dashed lines. The highly conserved active site glutamate residues Glu435 and Glu444, forming the double E-loop, bind to the substrate guanidinium group. The hydrogen bond (highlighted in yellow dashed lines) between the carbonyl oxygen of S1 and the amide nitrogen of G4 stabilizes the β-turn conformation. Abbreviation: ac, N-terminal acetylation.
Figure 29
Figure 29
PRMT7 (purple) from T. brucei bound to histone H4 peptide (gray) (PDB code 4M38). Polar contacts of <3.5 Å are represented as dashed lines.
Figure 30
Figure 30
Structural representation of the active site of rat PRMT1 (PDB code 1OR8), rat PRMT3 (PDB code 1F3L), rat PRMT4 (PDB code 3B3F), and mouse PRMT7 (PDB code 4C4A). All structures contain a bound cofactor (SAH, highlighted in gray). Abbreviation: Rsub, substrate arginine.
Figure 31
Figure 31
Structural comparison of the active site of rat PRMT1 bound to SAH and substrate arginine (PDB code 1OR8) and human PRMT5 bound to sinefungin and histone H4 peptide substrate (PDB code 4GQB).
Figure 32
Figure 32
Structural comparison of the active site pocket of C. elegans PRMT5 bound to SAH (PDB code 3UA3) and rat PRMT4 (CARM1) bound to SAH (PDB code 3B3F). The lower images depict the putative model of the enzyme active site pockets bound to its products, represented by SAH and SDMA in the case of PRMT5 or SAH and ADMA for PRMT4.
Figure 33
Figure 33
Proposed catalytic mechanism for type I PRMT enzymes, exemplified by PRMT1.
Figure 34
Figure 34
Mechanisms of lysine demethylation. (A) Active site of human LSD1 with bound FAD attached to the mechanism-based histone 3 peptide inhibitor N-methylpropargyl-K4 H3 (PDB 2UXN) and proposed mechanism. Note that the covalent inhibitor was further reduced using NaBH4. (B) Active site of the JMJD2A with bound H3K9me3, nickel, and N-oxalylglycine that both mimic the actual iron and α-ketoglutarate cofactors, highlighted in green and gray, respectively (PDB 2OQ6), and proposed mechanism. Green dashed lines represent CH···O hydrogen bonds. Abbreviations: FAD, flavin adenine dinucleotide; FADH, reduced flavin adenine dinucleotide; FA, formic acid; aKG, α-ketoglutarate; OGA, N-oxalylglycine.
Figure 35
Figure 35
Schematic overview of writers and readers of histone arginine methylation.
Figure 36
Figure 36
SMN Tudor domain bound to the asymmetric dimethylated arginine residue (PDB code 4A4G). The left panel illustrates the Tudor domain colored according to its electrostatic surface potential. The image on the right highlights the ADMA-interacting residues that form a hydrophobic cage around the methylated guanidinium group.
Figure 37
Figure 37
Nonselective PRMT inhibitors. Note that adenosine dialdehyde (22) and AMI-1 (25) are both not direct PRMT inhibitors. Adenosine dialdehyde blocks the activity of SAH hydrolase, which induces an increase in SAH levels, thereby inhibiting PRMT activity. AMI-1 binds to the histone substrates and prevents recognition by the PRMT enzyme.
Figure 38
Figure 38
SAM derivative AAI is transformed in situ to generate a bisubstrate PRMT inhibitor. The gray sphere denotes a peptidyl arginine substrate.
Figure 39
Figure 39
Bisubstrate-based PRMT inhibitors.
Figure 40
Figure 40
PRMT1-selective inhibitors.
Figure 41
Figure 41
Allosteric PRMT3 inhibitor SGC707. Crystal structure of dimeric PRMT3 bound to inhibitor 39 (PDB code 4RYL). SGC707 (gray) binds an allosteric site located at the interface between two PRMT3 protomers. Hydrogen bonds of <3.5 Å are represented as dashed black lines.
Figure 42
Figure 42
PRMT4 (CARM1)-selective inhibitors. The top panel illustrates the structural characterization of the indole inhibitor 40 bound to PRMT4 (PDB code 2Y1W). The lower image represents the crystal structure of PRMT4 bound to the pyrazole inhibitor 41 (PDB code 2Y1X). Dashed green lines indicate CH···O hydrogen bonds, whereas other polar contacts of <3.5 Å are represented as dashed black lines.
Figure 43
Figure 43
PRMT5-selective inhibitors.
Figure 44
Figure 44
Probes for PRMT1. The amidine group is highlighted in blue, whereas the reporter tags (biotin or fluorescein) are marked in red. IC50 values for the PRMTs were determined after incubation of the enzyme with 15 μM 14C-methyl-SAM for 10 min at 37 °C.
Figure 45
Figure 45
Nitroalkenes as cysteine-reactive PRMT1 inhibitors.
Figure 46
Figure 46
Arginine methylation sites in histone proteins. Abbreviations: a, asymmetric dimethylation; s, symmestric dimethylation; m, monomethylation. Color code: green, gene activation; red, gene repression; yellow, gene activation or repression, or unknown.
Figure 47
Figure 47
Protein arginine kinase McsB transfers the γ-phosphoryl group from ATP onto the arginine guanidinium group. The generated phosphoarginine residue can be hydrolyzed by the protein arginine phosphatase YwlE.
Figure 48
Figure 48
(A) Active site of l-arginine kinase with bound ADP, nitrate, and l-arginine (PDB code 1BG0) and proposed reaction mechanism. Note that several arginine residues were omitted in the proposed reaction scheme for clarity. (B) Active site of YwlE C7S with bound peptidyl arginine and containing a phosphorylated S7 residue, which mimics the thiophosphate reaction intermediate generated after the first SN2 reaction (PDB code 4KK4). Residue R149* originates from a symmetry-equivalent molecule. Polar contacts of <3.5 Å are represented as dashed lines. The proposed catalytic mechanism for the PAP enzyme YwlE is shown on the right side. The guanidinium group of the incoming phosphoarginine substrate is colored in blue, whereas the phosphoryl group is shown in red.
Figure 49
Figure 49
Generation of ADP-ribosylated arginines is catalyzed by ART enzymes, while the hydrolysis of peptidyl ADP-ribosylated arginine residues is mediated by ARH enzymes.
Figure 50
Figure 50
Post-translational addition of arginine residues is catalyzed by ATE1.
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