1 Structure of Fam20A reveals a pseudokinase featuring unique

2 disulfide pattern and inverted ATP-binding

3

4 Jixin Cui#,1, Qinyu Zhu#,2,3, Hui Zhang2,3, Michael A. Cianfrocco4, Andres E.

5 Leschziner4, Jack E. Dixon*1,4,5, Junyu Xiao*,3

6

7 1. Department of Pharmacology, University of California, San Diego, La Jolla, CA

8 92093, USA

9 2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing

10 100871, China

11 3. The State Key Laboratory of and Plant Research, School of Life

12 Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing,

13 100871, China

14 4. Department of Cellular and Molecular Medicine, University of California, San

15 Diego, San Diego, CA 92093, USA

16 5. Department of Chemistry and Biochemistry, University of California San Diego,

17 La Jolla, CA 92093, USA

18

19 # These authors contributed equally to this work

20 *Correspondence and requests for materials should be addressed to J.E.D

21 ([email protected]) and J.X. ([email protected])

1 22 Abstract

23 Mutations in FAM20A cause tooth enamel defects known as Amelogenesis

24 Imperfecta (AI) and renal calcification. We previously showed that Fam20A is a

25 secretory pathway pseudokinase and allosterically activates the physiological casein

26 kinase Fam20C to phosphorylate secreted important for biomineralization

27 (Cui et al., 2015). Here we report the nucleotide-free and ATP-bound structures of

28 Fam20A. Fam20A exhibits a distinct disulfide bond pattern mediated by a unique

29 insertion region. Loss of this insertion due to abnormal mRNA splicing interferes

30 with the structure and function of Fam20A, resulting in AI. Fam20A binds ATP in the

31 absence of divalent cations, and strikingly, ATP is bound in an inverted orientation

32 compared to other kinases. Fam20A forms a dimer in the crystal, and residues in the

33 dimer interface are critical for Fam20C activation. Together, these results provide

34 structural insights into the function of Fam20A and shed light on the mechanism by

35 which Fam20A mutations cause disease.

2 36 Introduction

37 The human kinome contains more than 500 members, which function

38 primarily in the cytoplasm or nucleus to regulate many cellular processes (Manning

39 et al., 2002). Approximately 10% of the kinome are considered pseudokinases due

40 to their lack of certain residues typically required for catalysis. Some of these

41 proteins, however, achieve activity using residues located at unconventional

42 positions and are thus bona fide kinases (Min et al., 2004, Eswaran et al., 2009, Villa

43 et al., 2009, Zhu et al., 2016). Others are completely devoid of catalytic activity but

44 play important scaffolding roles and can serve as allosteric regulators of active

45 kinases (Boudeau et al., 2006, Zeqiraj and van Aalten, 2010, Shaw et al., 2014, Kung

46 and Jura, 2016).

47 Novel kinases specifically residing in the secretory pathway, exemplified by

48 the family of sequence similarity 20 (Fam20) family, have been recently identified to

49 phosphorylate secreted and lumenal proteins and glycans (Tagliabracci et al.,

50 2013a, Tagliabracci et al., 2013b, Sreelatha et al., 2015). As commonly seen with

51 secretory proteins, these kinases are glycosylated and contain disulfide bonds for

52 structural integrity. Fam20C, the best-characterized member of the Fam20 family,

53 has been established as the physiological casein kinase that phosphorylates the vast

54 majority of the secreted phosphoproteome (Tagliabracci et al., 2012, Tagliabracci et

55 al., 2015). Fam20C substrates are involved in a wide spectrum of biological

56 processes, including the formation of bones and teeth (Tagliabracci et al., 2012, Cui

57 et al., 2015). Newborns with loss-of-function FAM20C mutations present with

58 deadly sclerosing osteomalacia with cerebral calcification known as Raine

3 59 syndrome (Simpson et al., 2007, Whyte et al., 2016). Patients with hypomorphic

60 FAM20C mutations can survive but manifest various anomalies, including tooth

61 enamel formation defect referred to as Amelogenesis Imperfecta (AI),

62 hypophosphatemia, and ectopic calcification (Fradin et al., 2011, Rafaelsen et al.,

63 2013, Takeyari et al., 2014, Acevedo et al., 2015, Elalaoui et al., 2016). The crystal

64 structure of the Fam20C ortholog from Caenorhabditis elegans (ceFam20) revealed

65 an atypical kinase architecture and key catalytic residues that are uniquely present

66 in the Fam20 family of kinases (Xiao et al., 2013).

67 Fam20B and Fam20A are two paralogs of Fam20C in vertebrates (Nalbant et

68 al., 2005). Fam20B phosphorylates a xylose residue during the biosynthesis of

69 proteoglycans and plays a key role in regulating glycan elongation (Koike et al.,

70 2009, Wen et al., 2014). Mutations of Fam20A result in AI and ectopic calcification

71 such as nephrocalcinosis, analogous to the defects caused by non-lethal Fam20C

72 mutations (Volodarsky et al., 2015, Cherkaoui Jaouad et al., 2015, Wang et al., 2014,

73 Wang et al., 2013, O'Sullivan et al., 2011, Kantaputra et al., 2014b, Kantaputra et al.,

74 2014a, Jaureguiberry et al., 2012, Cho et al., 2012, Cabral et al., 2013, Kantaputra et

75 al., 2017). We have previously demonstrated that Fam20A lacks an essential residue

76 for catalysis and is therefore the first pseudokinase identified in the secretory

77 pathway (Cui et al., 2015). Fam20A forms a functional complex with Fam20C and

78 allosterically increases Fam20C activity towards secretory substrates, including the

79 enamel matrix proteins, whose phosphorylation is critical for enamel formation (Cui

80 et al., 2015). As compared to other Fam20 family members, Fam20A has a unique

81 and highly conserved insertion in the Gly-rich loop (Xiao et al., 2013). Truncation of

4 82 this insertion due to aberrant RNA splicing causes AI, strongly suggesting its role in

83 maintaining Fam20A function (Cho et al., 2012). Furthermore, Fam20A binds ATP

84 despite being catalytically inactive (Cui et al., 2015), the structural basis and

85 functional impact of which remain elusive.

86 Here we report the nucleotide-free and ATP-bound crystal structures of

87 Fam20A. Although the kinase core of Fam20A is structurally similar to that of

88 ceFam20, Fam20A displays an unusual disulfide pattern dictated by a pair of

89 cysteine residues within the unique insertion region. Strikingly, ATP binds to

90 Fam20A in an unprecedented orientation independent of cations. These results

91 reinforce the conclusion that Fam20A is a pseudokinase in the secretory pathway

92 and facilitate a deeper understanding of AI caused by Fam20A mutations.

93

5 94 Results

95 Fam20A displays a unique disulfide pattern

96 We determined the crystal structure of human Fam20A at 2.5 Å resolution

97 (Figure 1A, Table 1). The kinase core of Fam20A (residues 160-525) is structurally

98 similar to that of ceFam20, and can be superimposed onto ceFam20 with a rmsd

99 (root-mean-square difference) of 2.1 A over 322 aligned Cα atoms. Most secondary

100 structures in the kinase core of Fam20A aligned well with the equivalent structural

101 elements in ceFam20 except for three regions: the Kβ1-Kβ2 loop, the Kβ6-Kβ7 loop,

102 and the Kβ3-Kα3 loop (hereafter we use “K” to denote the secondary structures in

103 the kinase core of the Fam20 proteins, and “N” to denote the secondary structures in

104 their unique N-terminal segments. Figure 1A, 1B; Figure 1-figure supplement 1). An

105 insertion of seventeen residues specifically exists in the Kβ1-Kβ2 loop of Fam20A

106 (Figure 1-figure supplement 1, Figure 1-figure supplement 2). This insertion forms

107 two short α-helices (Kα2A, Kα2B) and forces an upswing of the Kβ1-Kβ2 loop. The

108 Kβ1-Kβ2 loop in turn levers up the Kβ6-Kβ7 loop and induces the formation of a

109 short helix (Kα5A) in this region. The Kβ3-Kα3 loop also swings up to engage with

110 the Kβ6-Kβ7 loop. Gln258Fam20A, which replaces a Glu essential for the catalytic

111 activity of Fam20C (Glu213ceFam20), resides at the C-terminal end of the Kβ3-Kα3

112 loop. As a result of the upswing of this loop, Gln258Fam20A is dislodged from the

113 “active site” of Fam20A (Figure 1A). The N-terminal segment of Fam20A contains

114 two α-helices and adopts a different topology compared to that of ceFam20. A long

115 helix, Nα2, is held alongside the kinase core and is involved in binding to ATP as

116 described below.

6 117 Fam20A displays a distinct disulfide pattern compared to ceFam20. As we

118 previously described, four pairs of disulfide bonds exist in ceFam20 (Xiao et al.,

119 2013). Three of them are also present in Fam20A (Figure 1-figure supplement 1).

120 However, a disulfide bond corresponding to Cys273ceFam20-Cys277ceFam20 in the Kβ6-

121 Kβ7 loop is absent in Fam20A. Instead, Cys209Fam20A and Cys211Fam20A in the Kβ1-

122 Kβ2 loop form two disulfide bonds with Cys319Fam20A and Cys323Fam20A (the two Cys

123 that align with Cys273ceFam20 and Cys277ceFam20), respectively (Figure 1A, Figure 1-

124 figure supplement 1). These two disulfide bonds tether the Kβ1-Kβ2 loop to the

125 Kβ6-Kβ7 loop, imposing a strong restraint on the structural flexibility of this region.

126 Notably, Cys209Fam20A and Cys211Fam20A are within the 17-residue insertion in the

127 Kβ1-Kβ2 loop and are specifically conserved in Fam20A orthologs (Figure 1-figure

128 supplement 2), suggesting that this unique disulfide pattern of Fam20A is

129 evolutionarily conserved.

130 The functional importance of these two Fam20A-specific disulfide bonds is

131 further demonstrated by a patient-derived Fam20A splicing variant. The N-terminal

132 portion of the Kβ1-Kβ2 loop, including Cys209Fam20A and Cys211Fam20A, is encoded by

133 a small exon, exon 3, of the FAM20A gene. In an AI patient with compound

134 heterozygous mutations in FAM20A (Cho et al., 2012), one allele has an A-G

135 transition at the acceptor site of intron 2 (c.590-2A-G), resulting in exon 3 skipping.

136 The expressed Fam20A variant has amino acids 197-214 substituted with a Val

137 (Fam20A-Δexon3, Figure 1C). Since the other allele has a nonsense mutation

138 (R276X) that abrogates the majority of the kinase domain, Fam20A-Δexon3 is the

139 only form of Fam20A present in the patient that gave rise to the disease. To evaluate

7 140 the function of Fam20A-Δexon3, we co-expressed V5-tagged osteopontin (OPN)

141 with Flag-tagged Fam20A-WT or Fam20A-Δexon3 in U2OS cells, metabolically

142 labeled the cells with 32P orthophosphate, and analyzed incorporation of

143 radiolabeled phosphate into the V5-immunoprecipitates. Consistent with our

144 previous observation, ectopic expression of Fam20A-WT significantly enhanced the

145 kinase activity of endogenous Fam20C towards OPN, resulting in a higher level of

146 OPN phosphorylation (Figure 1D) (Cui et al., 2015). In contrast, expression of

147 Fam20A-Δexon3 failed to increase OPN phosphorylation. Compared with Fam20A-

148 WT, Fam20A-Δexon3 was poorly secreted and thus probably misfolded (Figure 1E).

149 Similarly, Fam20A-CC/AA, a Fam20A mutant having both Cys209Fam20A and

150 Cys211Fam20A mutated to Ala, also showed attenuated secretion and diminished

151 ability to enhance OPN phosphorylation (Figure 1F). Thus, the unique disulfide

152 pattern in Fam20A plays an important role in maintaining its structure and function.

153

154 Fam20A binds ATP without cations

155 We have previously demonstrated that both Fam20A and Fam20C could bind

156 ATP by means of thermal stability shift assays (Cui et al., 2015). Consistent with the

157 fact that metal ions such as Mn2+ are required for the kinase activity of Fam20C,

158 Fam20C bound ATP in the presence of Mn2+, whereas ATP alone induced little

159 changes in its melting temperature (Tm) (Figure 2A). In contrast, ATP alone caused

160 dramatic Tm increase of Fam20A, indicating that ATP binding to Fam20A could be

161 cation-independent (Figure 2A).

8 162 To determine the binding affinity of Fam20A/ATP interaction, we measured

163 the ΔTm of Fam20A at different ATP concentrations in the presence or absence of

2+ app 164 Mn , and deduced apparent dissociation constant (Kd ) values. This method has

165 been validated by Murphy et al. to evaluate the affinity of ATP for a number of

166 kinases (Murphy et al., 2014b). Our results show that Fam20A binds ATP with a

app 167 Kd of 3 μM in the absence of cations as compared to 156 μM in the presence of

168 saturating amount of Mn2+ (Figure 2B). This suggests that Fam20A preferably binds

169 ATP without cations.

170 Fam20A also binds other nucleoside triphosphates including GTP, CTP, and

171 UTP in the absence of metal ions, albeit to lesser degrees compared to ATP (Figure

172 2C). In contrast, Fam20C selectively binds Mn2+/ATP. Furthermore, Fam20A prefers

173 binding ATP over ADP (Figure 2D), indicating that the γ-phosphate group of ATP is

174 important for the interaction.

175 The significant Tm increase of Fam20A upon ATP binding suggests that ATP

176 stabilizes Fam20A structure. We wondered whether ATP also stabilizes the

177 Fam20A/Fam20C complex. Recombinantly purified Fam20A and Fam20C were

178 mixed (with Fam20A in slight excess to promote complex formation) and passed

179 through a gel filtration column (Figure 2E). In the absence of ATP, the peak for the

180 Fam20A/Fam20C complex appeared asymmetrical with a “shoulder”. When ATP is

181 present, the peak of the complex became much sharper, suggesting that ATP

182 improved the structural homogeneity of the Fam20A/Fam20C complex.

183 To further assess the effect of ATP on the assembly of the Fam20A/Fam20C

184 complex, we performed single particle electron microscopy on negatively stained

9 185 samples of Fam20A/Fam20C. Two-dimensional (2D) class averages of the complex

186 exhibited a clover-shaped structure that is consistent in size with a

187 Fam20A/Fam20C tetramer (Figure 2F). By comparing 2D class averages for each

188 condition, we observed that addition of ATP rendered the Fam20A/Fam20C

189 particles smaller in size (Figure 2F, Figure 2-figure supplement 1 and Figure 2-

190 figure supplement 2), indicating a more compact and less flexible conformation of

191 the complex. Collectively, these results suggest that ATP binding to Fam20A

192 stabilizes Fam20A as well as the Fam20A/Fam20C complex.

193

194 ATP binds to Fam20A in an inverted orientation

195 To elucidate how Fam20A binds ATP, we soaked ATP into the Fam20A

196 crystals obtained above and determined the complex structure at 2.75 Å resolution

197 (Figure 3A, Table 1). Surprisingly, ATP binds to Fam20A in an unusual orientation in

198 the structure. In an independent experiment, we also co-crystallized Fam20A with

199 ATP and determined the structure at 2.9 Å. The co-crystal structure is almost

200 identical to the soaked one, suggesting that the unexpected ATP-binding mode is not

201 an artifact caused by the soaking experiment.

202 In the Fam20A/ATP structure, the adenine moiety of ATP occupies a similar

203 pocket as that of ADP in the ceFam20/ADP structure; however, the entire nucleotide

204 is inverted in such a way that the ribose is turned “upside down”, and the phosphate

205 groups point to the opposite direction, reaching to the Nα2 helix (Figure 3B, 3C).

206 The adenine ring of ATP is sandwiched by several hydrophobic residues in Fam20A,

207 including Met235 and Phe343 from the top, and Pro279, Leu344, and Leu429 from

10 208 the bottom (Figure 3C). It also forms three hydrogen bonds with the side chain of

209 Ser341 and the main chain atoms of Ala342 and Leu344. The ribose is coordinated

210 by Arg352. The triphosphate group, especially the γ-phosphate, is surrounded by

211 extensive salt-bridge and hydrogen-bond interactions contributed by Tyr125 and

212 Lys129 from the Nα2 helix, as well as Lys233, Ser346, and the main chain amide

213 group of Leu347.

214 To confirm the inverted ATP-binding mode of Fam20A, we performed a

215 series of mutagenesis experiments. We have previously shown that the adenine

216 nucleotide binds to Fam20C in the conventional conformation, and is coordinated by

217 a number of conserved residues, including Lys271Fam20C and Asp478Fam20C (Xiao et

218 al., 2013). Lys271Fam20C is one of the two critical Lys that are involved in binding to

219 the phosphate groups of the adenine nucleotide. Asp478Fam20C aligns with the DFG

220 Asp in canonical kinases and is involved in binding to the metal ions that engage the

221 phosphate groups of ATP. Indeed, neither Fam20C-K271A nor Fam20C-D478A

222 mutant could bind ATP, as shown by the thermal stability shift assay (Figure 3D).

223 This is consistent with our previous result to show that these mutants have greatly

224 diminished kinase activity. In contrast, substitution of the corresponding residues

225 in Fam20A (Lys223Fam20A and Asp430Fam20A, respectively) with Ala has little effect

226 on the ability of Fam20A to bind ATP (Figure 3D). Furthermore, we generated

227 several Fam20A mutants to test the importance of residues involved in ATP-binding,

228 including Fam20A-K129A, Fam20A-K233A, Fam20A-M235W, and Fam20A-S346E.

229 Lys129Fam20A and Lys233Fam20A interact with the phosphate groups of ATP and help

230 maintain the ATP in the inverted orientation (Figure 3C). In addition, substituting

11 231 Met235Fam20A that is positioned near the ATP binding site with a bulkier Trp would

232 interfere with adenine binding. Finally, mutating Ser346Fam20A to a negatively

233 charged Glu would likely repel the phosphate groups of ATP. All four mutants

234 showed attenuated ATP binding as compared to the wildtype protein (Figure 3E).

235 Taken together, these mutation results corroborate our structural analyses,

236 demonstrating that Fam20A binds ATP in a novel, inverted conformation that is

237 fundamentally different from Fam20C and other kinases.

238

239 Dimerization of Fam20A

240 Fam20A dimerizes/oligomerizes in solution in a concentration-dependent

241 manner (Figure 4A). Also, V5-tagged Fam20A, but not Fam20B, co-

242 immunoprecipitated with Flag-tagged Fam20A when co-expressed in U2OS cells,

243 suggesting that Fam20A can form dimers/oligomers in the cell (Figure 4B). In the

244 crystal, Fam20A forms a symmetrical, face-to-face dimer (Figure 4C). Each molecule

245 in the dimer buries a ~2000 Å2 interface, which accounts for 9.5% of its total

246 solvent-accessible surfaces. The dimer is mediated by a number of hydrogen bonds

247 and hydrophobic interactions between the two protomers (Figure 4-figure

248 supplement 1). The Kβ1-Kβ2 loop from the two molecules facing each other, and the

249 two pairs of short helices Kα2A and Kα2B are involved in forming a small four-helix

250 bundle. The Kβ3-Kα3 loop of each protomer docks onto the C-lobe of the other

251 protomer in the dimer (Figure 4C). In particular, Val249, Phe251, and Phe254

252 protrude out of the Kβ3-Kα3 loop to make strong hydrophobic contacts with the

253 other molecule.

12 254 To determine whether the dimer interface seen in the crystal is important for

255 Fam20A function, we substituted Val249, Phe251 and Phe254 with Ala (Fam20A-

256 AAA). As demonstrated by the coimmunoprecipitation experiment, interaction

257 between Flag-tagged Fam20A-AAA and V5-tagged Fam20A-AAA was significantly

258 reduced compared to that between wild-type Fam20A (Figure 4B). Secretion of

259 Fam20A-AAA was not obviously changed, suggesting that the mutations did not

260 dramatically affect protein folding or stability (Figure 4D). Importantly, Fam20A-

261 AAA showed impaired interaction with Fam20C (Figure 4E). Consistently, it failed

262 to enhance Fam20C kinase activity in vitro (Figure 4F, 4G), nor could it increase

263 Fam20C-catalyzed OPN phosphorylation in the cell (Figure 4H). Taken together, our

264 results demonstrate that an intact dimer interface reported here is critical for

265 Fam20A to bind Fam20C and allosterically enhance Fam20C activity.

266

267 Discussion

268 The lumen of the endoplasmic reticulum as well as the Golgi apparatus is

269 oxidative in nature. In order to adapt to this environment, secretory pathway

270 proteins often form disulfide bonds to stabilize their structures. The crystal

271 structure of Fam20A reveals two unique disulfide bonds as compared to Fam20C,

272 which are mediated by a Fam20A-specific, evolutionarily conserved insertion.

273 Remarkably, loss of this insertion due to abnormal mRNA splicing caused AI in a

274 patient. Thus, compared to its close paralog Fam20C, Fam20A appears to have

275 “redesigned” its disulfide pattern to achieve a kinase-independent function.

13 276 It is not uncommon for a pseudokinase to bind ATP. A number of

277 pseudokinase structures have been determined with nucleotide bound, including

278 HER3, STRADα, ILK, RNase L, and the JH2 domains of the JAK kinases

279 (Bandaranayake et al., 2012, Fukuda et al., 2009, Han et al., 2014, Huang et al., 2014,

280 Jura et al., 2009, Min et al., 2015, Shi et al., 2010, Zeqiraj et al., 2009, Littlefield et al.,

281 2014). Among them, STRADα also binds ATP in a cation-independent manner.

282 Notably, like Fam20A, STRADα also utilizes positively charged residues to engage

283 the phosphate groups of ATP (Zeqiraj et al., 2009). This strategy could be used by

284 other pseudokinases that bind nucleotides without cations, such as MLKL (Murphy

285 et al., 2014b, Murphy et al., 2013, Murphy et al., 2014a). The Ca2+/calmodulin-

286 activated Ser-Thr kinase (CASK) also binds ATP without cations, although it appears

287 to possess some catalytic activity and function as an atypical kinase rather than a

288 pseudokinase (Mukherjee et al., 2008). The concentration of free cytoplasmic Mg2+

289 is on the order of 1 mM, and it is generally believed that ATP exists in the cell mostly

290 in a complex with Mg2+. However, these studies suggest that free ATP also plays a

291 role in various biological systems. A recent study has measured the cellular ATP and

292 Mg2+/ATP concentrations using 31P-NMR spectroscopy, and demonstrated that

293 these two forms of ATP are present at ~54 μM and ~400 μM in the cytosol,

294 respectively, and have a constant ratio of 0.135 (Gout et al., 2014). Although the

295 concentrations of Mg2+ (or Mn2+), ATP, and Mg2+/ATP (Mn2+/ATP) in the secretory

296 pathway are less clear, our results show that Fam20A can bind free ATP with a high

app 297 affinity (Kd ≈ 3 μM). Therefore, the divalent cation independent ATP-binding of

298 Fam20A is likely a physiologically relevant phenomenon.

14 299 What is unique about Fam20A is that it binds ATP in an inverted manner,

300 which is first seen for a kinase. With the phosphates pointing away from the kinase

301 domain, the Fam20A/ATP complex is unlikely catalytically competent. Nevertheless,

302 our results show that ATP binding stabilizes Fam20A structure and improves the

303 conformation homogeneity of the Fam20A/Fam20C complex. This is again

304 reminiscent of the pseudokinase STRADα, which requires the binding of ATP to

305 promote the assembly of the LKB1-MO25-STRADα complex and activate the kinase

306 activity of LKB1 (Zeqiraj et al., 2009). Although binding of ATP to Fam20A results in

307 few conformational changes in the crystal structures we reported here, the presence

308 of ATP greatly increases the melting temperature of Fam20A. It is likely that

309 without ATP, the structure of Fam20A is more flexible and less stable. Binding of

310 ATP may help stabilize the conformation of Fam20A by mediating additional

311 interactions between the kinase core and the N-terminal segment.

312 The Fam20A/Fam20C complex appears to be a tetramer that contains two

313 molecules of Fam20A and two molecules of Fam20C (Cui et al., 2015). Here we show

314 that Fam20A forms a dimer in the crystal, and residues in the dimer interface are

315 critical for Fam20A to bind and activate Fam20C. At this stage, it remains unclear

316 whether this Fam20A dimer also exists in the Fam20A/Fam20C tetramer. It is

317 possible that the Fam20A dimer seen here serves as a platform to allow the docking

318 of Fam20C and assembly of the complex. However, we cannot rule out the

319 possibility that the Fam20A dimer in the crystal is not physiological, and the surface

320 mediating Fam20A dimerization is actually used to interact with Fam20C directly. A

321 high-resolution Fam20A/Fam20C complex structure is ultimately needed to

15 322 elucidate how Fam20A interacts with Fam20C and allosterically regulates Fam20C

323 activity.

324 In conclusion, we have solved the crystal structure of Fam20A, a secretory

325 pathway pseudokinase. We have identified a novel mode of ATP binding and a

326 unique disulfide pattern that is indispensable for Fam20A function. Our results

327 expand the diversity of protein kinases and shed light on the molecular pathology of

328 diseases caused by mutations in Fam20A.

329

16 330 Materials and Methods

331 Protein Expression and Purification. DNA fragment encoding Human Fam20A

332 residues 69-529 was cloned into a modified pI-secSUMOstar vector (LifeSensors)

333 containing a tobacco etch virus (TEV) protease cleavage site. Bacmids were

334 generated using the Bac-to-Bac system (Invitrogen). Recombinant baculoviruses

335 were generated and amplified using the Sf21 insect cells (RRID: CVCL_0518),

336 maintained in the SIM SF medium (Sino Biological Inc.). For protein production, Hi5

337 cells (RRID: CVCL_C190) grown in the SIM HF medium (Sino Biological Inc.) were

338 infected at a density of 1.5-2.0 x 106 cells/ml. 48 hours post infection, 2 liters of

339 conditioned medium were collected by centrifugation at 200x g. The medium was

340 concentrated using a Hydrosart Ultrafilter (Sartorius) and exchanged into the

341 binding buffer containing 25 mM Tris-HCl, pH 8.0, 200 mM NaCl. The proteins were

342 then purified using the Ni-NTA resin (GE healthcare). The N-terminal 6xHis-SUMO

343 fusion tag was removed by the TEV protease. Untagged Fam20A was further

344 purified by the anion exchange chromatography using a Resource Q column (GE

345 healthcare), followed by the size-exclusion chromatography using a Superdex 200

346 increase 10/300 GL column (GE healthcare).

347

348 Thermal stability shift assay. For the thermal stability shift assays, proteins were

349 diluted to a final concentration of 2 μM in a buffer containing 20 mM HEPES, pH 7.5,

350 100 mM NaCl. Different concentrations of nucleotides or metal ions were added as

351 indicated. SYPRO Orange dye (Molecular Probes, S6650) was added to a final

352 concentration of 5× to monitor protein denaturation. Thermal scanning (25–85°C at

17 353 0.6°C/min) was performed using a LightCycler 480 System (Roche) with the

354 combination filters of 465 nm (excitation) and 580 nm (emission). Data were

355 analyzed using the software provided by the manufacture.

356

357 Crystallization. Fam20A in 20 mM HEPES, pH 7.5, 100 mM NaCl was concentrated

358 to 7 mg/ml and used for crystallization. The crystals were grown at 20°C using the

359 hanging-drop vapor-diffusion method. The precipitant solution contained 1.7 M

360 ammonium sulfate, 0.1 M HEPES, pH 7.5, and 6% (w/v) PEG 400. The crystals

361 reached full size in 10-14 days, and were then transferred into a cryo-protection

362 solution containing 20% (v/v) glycerol, 1.7 M ammonium sulfate, 0.1 M HEPES, pH

363 7.5, and 6% (w/v) PEG 400 and flash-frozen in liquid nitrogen. To obtain the ATP-

364 bound structure, both co-crystallization and soaking experiments were performed.

365 For co-crystallization, Fam20A protein solution was supplemented with 20 mM ATP

366 before mixed with above precipitant solution and crystallized. For soaking, the apo

367 crystal obtained above was soaked in the cryo-protection solution supplemented

368 with 10 mM ATP for 1 hour before frozen.

369

370 Data Collection and Structure Determination. Diffraction data were collected at

371 Shanghai Synchrotron Radiation Facility beamline BL17U and processed with

372 HKL2000 (HKL Research). The structure of Fam20A was determined by molecular

373 replacement using the ceFam20 structure as the search model in Phaser (McCoy et

374 al., 2007, RRID: SCR_014219). The structural model was then manually built in Coot

375 (Emsley et al., 2010, RRID: SCR_014222) and refined with Phenix (Adams et al.,

18 376 2010, RRID: SCR_014224). Five percent randomly selected reflections were used for

377 cross-validation (Brunger et al., 1998).

378

379 Bioinformatics and structural analysis. Multiple sequence alignment was

380 performed using PROMALS3D (Pei et al., 2008). Structural alignment between

381 Fam20A and ceFam20 was performed using Dali server (Holm and Rosenstrom,

382 2010, RRID: SCR_013433). Interaction between the two protomers in the Fam20A

383 dimer was analyzed using DIMPLOT (Laskowski and Swindells, 2011). Molecular

384 graphics were prepared using PyMol (Schrö dinger, LLC., RRID: SCR_000305).

385

386 Negative stain grid preparation & electron microscopy. FAM20A/20C

387 complexes were mixed with or without ATP and further purified using size

388 exclusion chromatography. After determining the concentration of peak fractions,

389 we diluted the sample in gel filtration buffer to 40 nM and incubated on continuous

390 carbon grids (Electron Microscopy Sciences, CF400-Cu) for 30 seconds. After

391 incubation, the grid was transferred directly onto 5x75 µl droplets of 2% uranyl

392 acetate and then blotted dry.

393 Negative stain grids were imaged on an Tecnai Sphera (FEI Company, Hillsboro, OR)

394 at 200 kV using a US4000 CCD detector (Gatan Inc., Pleaston, CA) with the Leginon

395 automated data collection software (Suloway et al., 2005) over a defocus range of 1

396 – 2.5 µm and a dose of 40 e-/Å2 at a nominal magnification of 62,000X (1.90 Å/pix).

397 93 micrographs were collected from the –ATP grid and 88 micrographs were

19 398 collected from the +ATP grid, representative micrographs are shown in Figure 2-

399 figure supplement 2.

400

401 Single particle image analysis. The Appion processing pipeline was used for

402 particle picking & extraction, as well as CTF estimation (Lander et al., 2009). For

403 each dataset, particles were initially picked using DoG Picker (Voss et al., 2009),

404 extracted at a pixel size of 3.80 Å/pix (64 x 64 pixels), and subjected to 2D

405 classification using Imagic (van Heel et al., 1996). From the resulting class averages,

406 representative class averages were used to re-pick particles using template-based

407 picking with FindEM (Roseman, 2004). The CTF for each micrograph was estimated

408 using CTFFIND4 (Rohou and Grigorieff, 2015). Particles were extracted from phase

409 flipped micrographs and binned to a pixel size of 3.80 Å/pix (64 x 64 pixels). This

410 yielded 6,051 particles for the “ATP-” dataset and 9,116 particles for the “ATP+”

411 dataset. 2D class averages shown in Figure 2-figure supplement 1 were calculated

412 from iterative rounds of 2D classification and alignment using Imagic (van Heel et

413 al., 1996) to finish with approximately 100 particles per 2D average.

414 For the alignment of “ATP-” and “ATP+” class averages in Figure 2F, SPIDER (Frank

415 et al., 1996) was used to find the highest cross correlation score between class

416 averages from each dataset. Following this comparison, the best matching pair were

417 aligned and shown in Figure 2F to illustrate the overall size difference between the

418 two datasets.

419

20 420 Others. In vitro kinase assay, mammalian cell culture, transfection,

421 immunoprecipitation, 32P orthophosphate metabolic labeling, as well as the

422 antibodies used in this study were described previously (Cui et al., 2015). U2OS

423 cells (RRID: CVCL_0042) were originally obtained from and authenticated by the

424 American Type Culture Collection (ATCC). Rabbit anti-V5 polyclonal antibody

425 (Millipore, AB3792; RRID: AB_91591) was used for immunoprecipitation.

426 Monoclonal anti-V5 antibody (Thermo Fisher Scientific, R960-25; RRID:

427 AB_2556564) and anti-FLAG antibody (Sigma, F3165; RRID: AB_259529) were used

428 for Western blotting.

429

430

21 431 Acknowledgments

432 We are grateful to staff members of Shanghai Synchrotron Radiation Facility 433 (beamline BL17U) for assistance in X-ray data collection. We thank Brenden Park 434 and Natalie Chen for their technical support, and Hongwei Guo for the access of the 435 Real-Time PCR machine. We thank Carolyn Worby, Xing Guo, Ping Zhang, and 436 members of the Xiao and Dixon laboratories for insightful discussions and 437 comments regarding the manuscript. 438 439 Funding

440 This work was supported by the National Science Foundation of China (31570735) 441 and the National Key Research & Development Plan (2016YFC0906000) to J.X., the 442 National Institutes of Health Grants (DK018849 and DK018024) as well as funds 443 from the Howard Hughes Medical Institute to J.E.D., and the Human Frontier Science 444 Program fellowship (LT000659/2013-L) to J.C.. M.A.C. is an HHMI Fellow of the 445 Damon Runyon Cancer Research Foundation (DRG 2171-13). 446 The funders had no role in study design, data collection and interpretation, or the 447 decision to submit the work for publication. 448 449

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29 696 Legends

697 Figure 1. The structure of Fam20A reveals a unique disulfide pattern.

698 (A) Structure of human Fam20A. The N-terminal segments were colored in green.

699 The Kβ1-Kβ2, Kβ6-Kβ7, and Kβ3-Kα3 loops were colored in red, teal, and magenta,

700 respectively. The rest of the kinase core is shown in white. The side chains of

701 Cys209, Cys211, Cys319, Cys323, and Gln258 are shown as sticks. The N- and C-

702 termini of the molecule are indicated. (B) Structure of ceFam20 (PDB ID: 4KQA) is

703 shown in the same orientation as Fam20A in (A) and colored in the same color

704 scheme. The side chains Cys273, Cys277, and Glu213 are shown as sticks. The

705 active site of ceFam20 is indicated with an oval in dashed line. (C) The schematic of

706 the human Fam20A gene structure and RNA splicing. Exon 3 is highlighted in red.

707 Relevant amino acid sequences are shown at the bottom. The two Cys residues in

708 the Kβ1-Kβ2 loop are highlighted in red. (D) Effect of Fam20A exon 3 deletion on

709 OPN phosphorylation. C-terminal V5-tagged OPN was expressed alone or co-

710 expressed with Flag-tagged Fam20A WT or Δexon3 in U2OS cells that were

711 metabolically labeled with 32P orthophosphate. OPN-V5 was immunoprecipitated

712 from the medium. Total OPN and 32P incorporation were detected by anti-V5

713 immunoblotting and autoradiography. Expression of Fam20A was monitored by

714 anti-Flag immunoblotting. (E) Effect of exon 3 deletion on Fam20A secretion. Flag-

715 tagged Fam20A WT or Δexon3 was expressed in U2OS cells and

716 immunoprecipitated from conditioned media using anti-Flag antibody. (F) Effect of

717 C209A/C211A mutation (CC/AA) on Fam20A secretion and OPN phosphorylation.

718

30 719 Figure 1-figure supplement 1. Sequence alignment of Fam20A, Fam20C, and

720 ceFam20 in the kinase core region.

721 The Kβ1-Kβ2, Kβ6-Kβ7, and Kβ3-Kα3 loops of the three proteins are highlighted in

722 red, teal, and magenta, respectively. The Cys residues are highlighted in orange.

723 Disulfide bonds conserved in Fam20A and ceFam20 are indicated with black dots

724 and lines. Disulfide bonds specific for Fam20A or ceFam20 are indicated with

725 orange dots and lines. The secondary structures of Fam20A and ceFam20 are

726 shown above and below the sequence blocks, respectively. Residues in Fam20A that

727 are involved in ATP-binding and dimer formation are highlighted with unfilled

728 rhombuses and triangles, respectively, at the top. Residues involved in binding to

729 Mn/ADP in ceFam20 are highlighted with filled gray rhombuses at the bottom.

730

731 Figure 1-figure supplement 2. Sequence alignment of Fam20A, Fam20B, and

732 Fam20C family members around the Fam20A-specific insertion region.

733 Conserved residues are highlighted with black rectangles. The Fam20A-specific

734 insertion is highlighted using a red rectangle, and the two conserved Cys are shaded

735 in red. The secondary structures of Fam20A are shown above the sequence blocks

736 and labeled.

737

738 Figure 2. Fam20A binds ATP without cations.

739 (A) Thermal stability shift assays of ATP binding to Fam20A or Fam20C in the

2+ 740 presence or absence of Mn . ATP, 250 μM; MnCl2, 1 mM. (B) Fam20A thermal

741 stability shift assay. The ΔTm values are plotted against ATP concentration. Data

31 742 points are represented as mean ± SD and fitted by non-linear regression of the

743 equation Y = ΔTm max*X/(Kd + X). (C) Thermal stability shift assays of ATP, GTP,

744 CTP or UTP binding to Fam20A or Fam20C. Nucleotide concentration is 10 μM for

745 Fam20A and 250 μM for Fam20C. (D) Thermal stability shift assays of ATP or ADP

746 binding to Fam20A without cations. Data were plotted and fitted as in (B). (E) Gel

747 filtration analyses of the Fam20A/Fam20C complex in the absence or presence of

748 250 μM ATP. (F) 2D class averages of the negatively stained Fam20A/20C

749 complexes. Representative 2D class averages of the complex assembled in the

750 presence or absence of ATP (250 μM) are shown at the same pixel size to highlight

751 the overall size changes between the two samples. Scale bar, 200 Å.

752

753 Figure 2-figure supplement 1. 2D class averages of the Fam20A/20C complex

754 assembled in the presence or absence of ATP (250 μM) determined by negative-

755 stain single particle electron microscopy. Scale bar, 200 Å.

756

757 Figure 2-figure supplement 2. Representative negative stain micrographs of the

758 Fam20A/20C complex assembled in the presence or absence of ATP (250 μM). Scale

759 bar, 200 nm.

760

761 Figure 3. ATP binds to Fam20A in an inverted orientation.

762 (A) The Fo-Fc difference electron density map (calculated before ATP is built and

763 contoured at 3σ) is shown as a green mesh, revealing the presence of ATP. Fam20A

764 is shown as ribbons and colored in the same scheme as Figure 1A (the Kβ1-Kβ2 loop

32 765 in red). The ATP molecule built afterwards is shown in sticks, and its carbon atoms

766 are colored in magenta. (B) Superimposition of Fam20A/ATP and ceFam20/ADP.

767 Fam20A and ceFam20 are shown in white and cyan, respectively. The carbons in

768 ATP and ADP are colored in magenta and cyan, respectively. (C) An enlarged image

769 of the ATP-binding pocket of Fam20A showing the detailed molecular interactions

770 with ATP. Fam20A is colored in the same scheme as in Figure 1A, with the Nα2

771 helix highlighted in green. Salt-bridge and hydrogen-bond interactions are shown

772 as dashed lines. (D) Thermal stability shift assays of ATP binding to Fam20C or

773 Fam20A. 100 μM and 250 μM ATP were used to assay Fam20A and Fam20C,

774 respectively. 5 mM MnCl2 was present in the Fam20C assay conditions. (E) Thermal

775 stability shift assays of ATP binding to Fam20A WT, K129A, K233A, S346E, and

776 M235W. Data were plotted and fitted as in Figure 2B.

777

778 Figure 4. Fam20A forms a dimer in the crystal and an intact dimer interface is

779 essential for its function.

780 (A) Fam20A dimerizes in solution in a concentration-dependent manner. 1 mg or

781 100 μg purified Fam20A (both in 500 μl) were individually loaded on a Superdex

782 200 increase 10/300 GL column (GE healthcare) and eluted using 20 mM HEPES, pH

783 7.5, and 100 mM NaCl. The peak positions of the two runs are highlighted with

784 dashed lines. The elution volumes of the molecular weight standards are indicated.

785 (B) Co-immunoprecipitation of Fam20A-Flag and Fam20A-V5 (or Fam20B-V5) from

786 U2OS cells. AAA, Fam20A V249A/F251A/F254A mutant. (C) The structure of

787 Fam20A dimer. One protomer in the dimer is shown in ribbons, and the other is

33 788 shown in surface representation. The Kβ1-Kβ2 and Kβ3-Kα3 loops are highlighted

789 in red and magenta, respectively. The rest of the two molecules are shown in white

790 and cyan. A close view of the Kβ3-Kα3 loop mediated interaction is shown on the

791 right. The side chains of Val249, Phe251, Phe254 are shown as sticks. (D) Secretion

792 of Fam20A WT and V249A/F251A/F254A. (E) Effect of V249A/F251A/F254A

793 mutation on Fam20A-Fam20C interaction. Fam20A-V5 (WT or AAA) and Fam20C-

794 Flag were co-expressed in U2OS cells and coimmunoprecipitated from the cell

795 lysate. (F and G) Effect of Fam20A-AAA on ENAM phosphorylation by Fam20C in

796 vitro. (H) Effect of Fam20A-AAA on OPN phosphorylation in metabolically 32P

797 radiolabeled U2OS cells.

798

799 Figure 4-figure supplement 1. Schematic drawing of the interactions between the

800 two protomers in the Fam20A dimer. Residues involved in forming hydrophobic

801 interactions are indicated, and the hydrophobic contacts are represented by spoked

802 arcs. Residues participating in hydrogen bonds are shown in ball-and-stick

803 representation, and the hydrogen bonds are shown as green dashed lines. Note that

804 the Fam20A dimer is symmetrical, therefore two sets of interactions shown in the

805 picture are present in the dimer structure.

806

34 807 Table 1. Data collection and refinement statistics. 808 809 Fam20A Fam20A with ATP (PDB ID: 5WRR) (PDB ID: 5WRS) Data collection Space group P 32 2 1 P 32 2 1 Cell dimensions a=156.854 Å, a=157.202 Å, b=156.854 Å, b=157.202 Å, c=143.655 Å c=144.802 Å Wavelength (Å) 0.979 1.009 Resolution (Å) 2.50 2.75

Rmerge 0.172 (1.804) 0.168 (1.470) Rpim 0.055 (0.566) 0.053 (0.472) CC1/2 (highest-resolution shell) 0.841 0.788 I / σI 28.8 (2.8) 21.2 (2.4) Completeness (%) 99.9 (100.0) 99.9 (100.0) Multiplicity 10.9 (11.1) 10.7 (10.6) Wilson B-factor 54.1 57.9

Refinement Reflections used in refinement 70216 (6906) 53997 (5329)

Reflections used for Rfree 1994 (197) 1946 (193) Rwork / Rfree 0.215 / 0.231 0.203 / 0.238 No. of non-hydrogen atoms Protein 7039 7018 Ligands 98 146 Protein residues 876 874 B-factors Protein 75.1 72.3 Ligands 116.7 101.4 R.m.s deviations Bond lengths (Å) 0.002 0.003 Bond angles (°) 0.62 1.01 Ramachandran Favored (%) 96.8 96.5 Allowed (%) 3.1 3.5 Outliers (%) 0.1 0 810 Each dataset was collected from a single crystal. Values in parentheses are for highest-resolution shell.

35 A Kβ1−Kβ2 Kβ6−Kβ7 B C319 C209 C323 Kβ3−Kα3 N C273 C277 C211 Nα2 Q258 E213

N

C C

C Genomic DNA 2 3 4

Spliced mRNA

D197YSQDEKALLGACDCTQI214 V197 Protein WT Δexon3

D OPN-V5 E F OPN-V5 20A-Flag 20A-Flag WT CC/AA WT Δexon3 20A-Flag WT Δexon3 autorad V5 IP from autorad Flag IP from medium α-V5 medium (OPN)

medium α-V5 V5 IP from V5 IP (OPN) Flag IP from

WB: α -Flag medium Lysate α-Flag α-Flag Lysate (20A) (20A) Lysate

Figure 1 Kα1 Kα2 Kβ1 Kα2A Kα2B Fam20A ss Fam20A 160 SWVQFHLGINRHGLYSRSSPVVSKLLQDMRHFPTISADYSQDEKALLGACDCTQIVKPSGVH Fam20C 225 NWLKFHIGINRYELYSRHNPAIEALLHDLSSQRITSVAMKSG------GTQ ceFam20 133 NWERFYSNIGSCSVYSD-DQMIDNLLHDLNTSPIKHVHIMDG------GTQ ceFam20 ss

Kβ2 Kβ3 Kα3 Fam20A ss Fam20A 222 LKLVLRFSDFGKAMFKPMRQQRDEETPVDFFYFIDFQRHNAEIAAFHLDRILDFRRVPPTVG Fam20C 270 LKLIMTFQNYGQALFKPMKQTREQETPPDFFYFSDYERHNAEIAAFHLDRILDFRRVPPVAG ceFam20 177 VKFVFTFKNDKQAVFKPMRFGRDYESDPNHFYFSDFERHHAEIATFHLDRVLGFRRAIPTVG ceFam20 ss

Kβ4 Kα4 Kα5 Kβ5 Kβ6 Kα5A Kβ7 Fam20A ss Fam20A 284 RIVNVTKEILEVTKNEILQSVFFVSPASNVCFFAKCPYMCKTEYAVCGNPHLLEGSLSAFLP Fam20C 332 RMVNMTKEIRDVTRDKKLWRTFFISPANNICFYGECSYYCSTEHALCGKPDQIEGSLAAFLP ceFam20 239 RVLNMTTELFEKA-EKKLKKTFFFSPAKNFCFVSRCDYYCDTTHAICGLPDMKEGSVQVFLP ceFam20 ss

Kβ8 Kα6 Kα7 Fam20A ss Fam20A 346 SLNLAPRLSVPNPWIRSYTLA-GKEEWEVNPLYCD-TVKQIYPYNNSQRLLNVIDMAIFDFL Fam20C 394 DLSLAKRKTWRNPWRRSYHKR-KKAEWEVDPDYCE-EVKQTPPYDSSHRILDVMDMTIFDFL ceFam20 300 DESAVPRKHNRSPYRRTYSKKNQVAEWQSSMNYCTDKVKTKRQYAHGRRLLDLVDIHILDYL ceFam20 ss

Kβ9 Kα8 Kα9 Fam20A ss Fam20A 406 IGNMDRHHYEMFTKFGD-DGFLIHLDNARGFGRHSHDEISILSPLSQCCMIKKKTLLHLQLL Fam20C 454 MGNMDRHHYETFEKFGN-ETFIIHLDNGRGFGKYSHDELSILVPLQQCCRIRKSTYLRLQLL ceFam20 362 IGNQDRHHFESFNVFNDLPSYAIHLDHGRAFGRSDFDDDDIILPLRQCCILRPSTFQTLMNF ceFam20 ss

Kα10 Kα11 Kα12 Fam20A ss Fam20A 467 AQADYRLSDVMRESLLEDQLSPVLTEPHLLALDRRLQTILRTVEGCIVAHGQQSVIVDG Fam20C 515 AKEEYKLSLLMAESLRGDQVAPVLYQPHLEALDRRLRVVLKAVRDCVERNGLHSVVDDD ceFam20 424 YSTPKSLTKALHESLSKDPAHPILAYKHYPAMERRLAKIMSHILECFESRGVAEVLVAE ceFam20 ss

Figure 1-figure supplement 1 Kα2 Kβ1 Kα2A Kα2B Kβ2

H.sapiens SPVVSKLLQDMRHFPTISADYSQDEKALLGACDCTQIVKPSGVHLKLVLRF M.musculus SLAISKLLHDMRHFPTISADYSQDEKALLGACDCSQIVKPSGVHLKLVLRF Fam20A R.norvegicus SPVVSKLLHDMRHFPTISADHSQDEKALLGACDCSQIVKPSGVHLKLVLRF G.gallus SPAVSRLLRDMHDFATISADYSQDEKALLGACDCSQIVKPSGVHLKLVLRF D.rerio DSSVDTLLQDMQTDTVINADFTQDEKALKGACDCSQVVKPSGLHLKLALKL H.sapiens TPELGAVMHAMATKKIIKADVGYK------GTQLKALLIL M.musculus TPELGAIMHAMATKKIIKADVGYK------GTQLKALLIL R.norvegicus TPELGAVMHAMATKKIIKADVGYK------GTQLKALLTL Fam20B G.gallus TPELGAVMHAMSTKKIVKADVGYK------GTQLKALLIL D.rerio TPELAAVLNALATAHVERADVGYK------GTQLKALLVL D.melanogaster DSSMGQLLETLRREPITRVSNLGR------GTQLKLLVRL H.sapiens NPAIEALLHDLSSQRITSVAMKSG------GTQLKLIMTF M.musculus NPAIDALLRDLGSQKITSVAMKSG------GTQLKLIMTF R.norvegicus NPAIDALLHDLGSQKITSVAMKSG------GTQLKLIMTF Fam20C G.gallus DPLMPTLLQDLATMKIVSSVQKSG------GTQLKLIMTF D.rerio NPVITALLRDLLSQKISSVGMKSG------GTQLKLIMSF D.melanogaster DTLVDAVLRDMIKLPIQHVVQKEG------GTQLKLIIEY C.elegans DQMIDNLLHDLNTSPIKHVHIMDG------GTQVKFVFTF

Figure 1-figure supplement 2 15 12 20A 20C A 20A B C 8 6 20C ) ) no cation

C 8 10 C °

ATP ° (

( Mn

(°C ) m

ATP-Mn m 4 3 T T m T 5 4

0 0 0 0 0 50 100 P P P P Mn no cation T T T T ATP conc. (µM) ATP GTP C U ATP GTP C U

12 D E 20A/C F

) 30 30

C 8 u ° (

A 20A/C 20A ATP - m m 20A T 15 15 4 ATP ADP ATP + 0 0 0 0 50 100 10 15 ml 10 15 ml ATP/ADP conc. (µM) ATP - ATP +

Figure 2 ATP -

ATP +

Figure 2-figure supplement 1 ATP - ATP +

Figure 2-figure supplement 2 A C Nα2

ATP K233 M235 K129

F343 S341 A342 ATP S346 B Y125 P279 L347 L429 L344 R352

ATP E ADP 10 9 6 5 WT 3 WT K129A K233A

12 ) 0 0 C °

( 0 50 100 0 50 100 D m T

) 10 12 C ° ( 6 m

T 8 WT WT M235W 5 S346E 4 0 20C-WT 20A-WT 0 0 20C-K271A 20A-K223A 0 50 100 0 50 100 20C-D478A 20A-D430A ATP conc. (µM)

Figure 3 A B 20B-V5 + 20A-V5 WT WT AAA AAA 20A-Flag WT WT WT AAA AAA

α-Flag

IP: α-V5 α-V5

Lysate α-Flag C

V249

F254 F251

D E F 20A-V5 WT AAA

20A-V5 AAA WT 20C-Flag + + + 20C + 20C + 20C 20A-WT 20A-AAA α-Flag V5 IP from IP: Time (min) 0 2 5 0 2 5 0 2 5 medium α-V5 α -V5 α-V5 autorad ENAM Lysate WB: (173-277) Lysate α-Flag Coomassie G H OPN-V5

20A-Flag AAA 20A - WT AAA WT

autorad 20C + + + + + + A:C ratio - 1 1 2 5 10 α-V5 medium

autorad from V5 IP (OPN) ENAM α-Flag (173-277) Coomassie Lysate (20A)

Figure 4 Mol A/B H413 I360 E415 E442 P358 L353 V355 P356 Y377 T380 I384 M409 A204

C323 A204 F251 Y253 C211 V249 F254 Y363 T212 V215 R361 T364 Mol B/A

Figure 4-figure supplement 1