Peptidylarginine deiminase 4 PAD4,,, Structural basis for Ca 2+ -induced activation of peptidylarginine deiminase 4 (PAD4) and histone N-terminal recognition Kyouhei ARITA, Toshiyuki SHIMIZU, Hiroshi HASHIMOTO, Mamoru SATO International Graduate School of Arts and Sciences, Yokohama City University DNA H2A, H2B, H3, H4 (H2A-H2B) 2 (H3) 2 (H4) 2 H H2A, H2B, H3, H4 N Peptidylarginine deiminase 4 PAD4 Ca 2+ (Fig. 1) PAD 5 PAD1-4, PAD6 PAD4 nuclear localization signal: NLS [1] PAD [2] PAD4 (Fig. 1)[3, 4] H3 Arg17 H4 Arg3 CARM1 (cofactor associated arginine methyltransferase 1) PRMT1 (protein arginine methyltrasnferase 1) [5, 6] PAD4 H3 Arg2, Arg8, Arg17, Arg26 H4 Arg3 H3 Arg17 H4 Arg3 CARM1 PRMT1 CARM1 PRMT1 PAD4 (Fig. 1)[3, 4] PAD4 PAD 1) PAD4 [7] 2) PAD PAD 3) MHC II (HLA-DRB1*0401) 4) PAD [8] PAD4 PAD4 PAD4 PAD4 [9] [10]
Figure 1 Transcriptional regulation by histone arginine citrullination by PAD4 and methylation by CARM1 and PRMT1. PAD4 Ca 2+ PAD4 0.1 M Imidazole ph 8.0, 0.2 M Li 2 SO 4, 8 % PEGMME2000 [11] EMTS Ethyl mercurithiosalicylate), TMLA(Trimethyl lead acetate), KAu(CN) 2, K 2 PtCl 4, LuCl 3 MIRAS Ca 2+ PAD4 Ca 2+ PAD4- benzoyl L-arginine amide: BA PAD4 C645A Ca 2+ Ca 2+ BA PAD4 C645A [Ca 2+ PAD4(C645A)] Ca 2+ PAD4 PAD4- Ca 2+ PAD4 C645A Ca 2+ H3 Arg8 H3 Arg17 H4 Arg3 Table 1 Fig. 2a PAD4 PAD4 2 N (N-terminal domain) C (C-terminal domain) Ca 2+ Ca 2+ PAD4 Ca 2+ PAD4- BA 5 3 N 2 Table 1. Crystallographic data, and data collection and refinement statistics Ca 2+- free Ca 2+ -bound BA Peptide H3-1 Peptide H3-2 Peptide H4 PAD4 PAD4 Complex Complex Complex Complex Crystallographic data and data collection statistics Space group C2 C2 C2 C2 C2 C2 Cell dimensions a (Å) 144.6 146.4 146.1 146.3 146.1 146.2 b (Å) 60.4 60.1 60.0 60.8 60.1 60.6 c (Å) 113.4 115.3 115.4 115.1 115.7 115.2 ß ( ) 123.6 124.4 124.2 124.3 124.3 124.2 Resolution range (Å) 33.42-2.80 50.00-2.60 50.00-2.30 50.00-2.00 50.00-2.07 50.00-2.25 Total observations 93,345 127,227 273,837 233,822 222,220 153,298 Unique reflections 38,041 24,864 36,718 55,675 47,513 39.724 Completeness (%) 97.6 (98.3) 96.2 (96.8) 97.9 (93.5) 97.7 (96.2) 92.6 (63.9) 98.3 (98.2) R merge (%) 4.6 (26.1) 5.0 (36.1) 5.0 (21.7) 5.6 (39.5) 6.2 (33.3) 6.1 (34.5) Refinement Resolution (Å) 2.80 2.60 2.30 2.10 2.10 2.25 No. reflections 17,603 22,280 32,646 43,126 41,436 35,325 R /R free (%) 23.1 / 26.4 22.4 / 26.9 21.1 / 25.2 20.2 / 24.1 20.2 / 24.6 19.9 / 24.8 No. atoms Protein 4,382 4,745 4,951 4,952 4,937 4,943 Substrate 0 0 20 39 40 37 Ca 2+ 0 5 5 5 5 5 Water 0 64 162 224 191 154 R.m.s. deviations Bond lengths (Å) 0.019 0.017 0.014 0.012 0.012 0.012 Bond angles ( ) 1.894 1.662 1.595 1.465 1.468 1.622
Figure 2 Overall structure of the substrate complex of Ca 2+ -bound PAD4 (C645A). Ribbon representation of the monomeric form of the substrate complex. Five Ca 2+ ions (Ca1, Ca2, Ca3, Ca4, and Ca5) are shown as black balls, and the substrate, benzoyl-l-arginine amide (BA), as a dark blue ball-and-stick model. Sub-domains 1 and 2 and the C-terminal domain are presented in yellow, green, and red, respectively. The putative nuclear localization signal (NLS) region is shown by a dotted line. Ribbon representation of the dimeric form of the substrate complex. A crystallographic 2-fold axis runs vertically at the center of the dimer. N 1-300 2 sub-domain 1 sub-domain 2 (Fig. 2a) sub-domain 1 sub-domain 2 sub-domain1 56 PPAKKKST 63 NLS 2 β-strands disorder G55S, V82A, G112A sub-domain 1 C G55S, V82A, G112A Ca 2+ 2 PAD4 sub-domain 2 3 Ca 2+ (Fig. 2a) Ca 2+ PAD4 disorder Ca 2+ PAD4 Ca 2+ PAD4- Ca 2+ α-helix (Fig. 3b) Ca 2+ Asp388 153 179 Asp155 Asp157 Asp176 Ca 2+ Ca 2+ Protein kinase C (PKC) C2 PKC C2 Ca 2+ sub-domain 2 Ca 2+ Figure 3 Structures around substrate and Ca 2+ binding sites in Ca 2+ -bound PAD4 (C645A). The substrate, benzoyl-l-arginine amide (BA), is shown as a green stick model. Ca 2+ ions are shown as yellow balls. Structure around Ca1, Ca2, and the substrate, benzoyl-l-arginine amide, in the C-terminal domain. Structure around Ca3, Ca4, and Ca5 in the N-terminal domain. C 5 Ca 2+ EF (Fig. 3) Ca 2+ BA C 2 Ca 2+ PAD4 2 1 (Fig. 2b) 2 PAD4 2 2 301-663 C Fig. 2a α/β 5 5 ββαβ module Fig. 4α/β L- L- arginine deiminase (ADI)[12] L- guanidinoacetic acid arginine:glycine amidinotransferase (AT)[13] L- α/β BA 1 Ca 2+ (Ca1) 1 Ca 2+ (Ca2) 2 ββαβ modules 2, Fig. 4 α-helix β-strand Ca1 Gln349, Glu353, Glu411 Phe407 Leu410 2 Ca2 Glu351, Asp369, Asn373 Ser370 1 (Fig. 3a) Fig. 3a BA BA 2
Asp350, His471, Asp473, Cys645 4 PAD4 Figure 4 Ribbon representation of the C-terminal domain in the substrate complex of Ca 2+ -bound PAD4 (C645A). Five ββαβ modules 1, 2, 3, 4, and 5 are colored light blue, red, dark blue, yellow, and orange, respectively. The substrate, benzoyl-l-arginine amide (BA), is shown as a green stick model. Ca 2+ ions are shown as yellow balls. Asp350 Asp473 Cys645(Ala645) His471 (-CH 2 -CH 2 -CH 2 -) Trp347 Val469 BA BA Arg374 Arg639 BA Arg374 PAD4 L- BA L- PAD4 BA (Asp350, His471, Asp473, Cys645) L L ADI Asp350, His471, Asp473, Cys645 PAD Ca 2+ PAD4, Ca 2+ PAD4 Ca 2+ PAD4- BA PAD4 Ca 2+ Ca 2+ PAD4 disorder acidic concave surface (Fig. 5a) 2 Ca 2+ (Ca1 Ca2) disorder (active site cleft) (Fig. 5b) Ca 2+ PAD4 Ca 2+ PAD4-BA 2 Ca 2+ (Ca1 Ca2) BA (Fig. 5c) 2 Ca 2+ (Ca1 Ca2) Ca1 Ca2 PAD E351A PAD N Ca 2+ C Asp388 Ca3 PAD4 Ca1 Ca2 PAD4 Ca 2+ Ca 2+ (active site cleft) Ca 2+ (c) Figure 5 Electrostatic surface potentials of Ca 2+ -free PAD4, Ca 2+ -bound PAD4, and the substrate(ba) complex of Ca 2+ -bound PAD4 (C645A) (c). Surface colors represent the potential from -10 k B T -1 (red) to +10 k B T -1 (blue). The substrate, benzoyl-l-arginine amide (BA), is shown as a green space-filling model. The acidic concave face and the active site cleft are marked by green and yellow circles, respectively.
Calpain[14] Lys Gln Transglutaminase[15] Ca 2+ Calpain PAD4 disorder Ca 2+ PAD4 BA PAD4 PAD4 Ca 2+ PAD4 C645 N 10 N 3 H3 1 Ac- 4 KQTARKSTGG 13 peptide H3-1 H3 2 Ac- 14 KAPRKQLATK 23 peptide H3-2 H4 Ac- 1 SGRGKGGKGL 10 peptide H4 3 N N (N-1), (N-2), (N-3), C (N+1), (N+2), (N+3) (Figs.6, 7b) 10 (N-2) (N+2) 5 (N-2) (N+1) 4 PAD4 (Fig. 6) (N-2) (N+1) 4 (N-2) H3-2 (N-2) Ala (c) Figure 6 Structures around the active sites of the Ca 2+ -bound PAD4 (C645A) in complex with peptides H3-1, H3-2, and H4. Left, ball-and-stick representation of the structures. The protein moiety is colored grey, and the peptides, H3-1, H3-2, and H4 (c) are colored green, magenta, and yellow, respectively. Superimposed are F o F c electron densities of the peptides, contoured at 2σ. Right, schematic diagrams of the structures on the left. Dotted lines and green half-circles show hydrogen bonds and hydrophobic interactions, respectively.
(N-2) PAD4 (N-2) PAD4 5 (N-2) Gly, Ala, Ser β (Fig. 7a) (N-2) (N+2) 5 (N-2) (N+1) 4 PAD4 (Fig. 6) (N+2) PAD4 PAD4 Arg374 β (Fig. 7b) (N-2) (N+2) (N+2) β (N-1) (N+2) β, N PAD4 β PAD4 PAD4 [16] PAD4 PAD4 β PAD4 PAD4 N PAD4 PAD4 Figure 7 Histone N-terminal structures. structural comparison of PAD4-bound forms. Peptides H3-1, H3-2, and H4 are shown as ball-and-stick representations colored green, magenta, and yellow, respectively. top view of the peptide H3-2 structure shown in, together with a molecular surface representation near the active site cleft. The weak intra-peptide interactions between the backbone oxygen at (N-1) position and the backbone nitrogen at (N+2) position are shown as dotted line. Ca 2+ PAD4, Ca 2+ PAD4, Ca 2+ PAD4 3 C Ca 2+ Ca 2+ Ca 2+ N PAD4 β PAD4, PAD4 PAD4-
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