Accepted Manuscript HDX Reveals Unique Fragment Ligands for the Vitamin D Receptor Matthew W Carson, Jun Zhang, Michael J Chalmers, Wayne P. Bocchinfuso, Karol D. Holifield, Thierry Masquelin, Ryan E Stites, Keith R Stayrook, Patrick R Griffin, Jeffery A. Dodge PII: DOI: Reference:

S0960-894X(14)00579-4 http://dx.doi.org/10.1016/j.bmcl.2014.05.070 BMCL 21684

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

2 April 2014 16 May 2014 20 May 2014

Please cite this article as: Carson, M.W., Zhang, J., Chalmers, M.J., Bocchinfuso, W.P., Holifield, K.D., Masquelin, T., Stites, R.E., Stayrook, K.R., Griffin, P.R., Dodge, J.A., HDX Reveals Unique Fragment Ligands for the Vitamin D Receptor, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.070

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HDX Reveals Unique Fragment Ligands for the Vitamin D Receptor. Matthew W Carsona*, Jun Zhangb, Michael J Chalmersb,c, Wayne P. Bocchinfusoa , Karol D. Holifielda, Thierry Masquelina, Ryan E Stitesa, Keith R Stayrooka, Patrick R Griffinb,c, Jeffery A. Dodgea a

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285

b

Department of Molecular Therapeutics

c *

The Scripps Research Molecular Screening Center (SRMSC)

Correspondence: [email protected]

Abstract- Modulation of the vitamin D receptor (VDR) with a ligand has the potential to be useful for the oral treatment of osteoporosis. One component of our lead generation strategy to identify synthetic ligands for VDR included a fragment based drug design approach. Screening of ligands in a VDR fluorescence polarization assay and a RXR/VDR conformation sensing assay resulted in the identification of multiple fragment hits (lean > 0.30). These fragment scaffolds were subsequently evaluated for interaction with the VDR ligand binding domain using hydrogen-deuterium exchange (HDX) mass spectrometry. Significant protection of H/D exchange was observed for some fragments in helixes 3, 7, and 8 of the ligand binding domain, regions which are similar to those seen for the natural hormone VD3. The fragments appear to mimic the A-ring of VD3 thereby providing viable starting points for synthetic expansion.

Graphical Abstract

The vitamin D receptor (VDR) is a ligand activated cofactor and a member of the nuclear hormone receptor family.1 VDR is naturally activated by the secosteroidal ligand vitamin 1,25 (OH)2-D3 or VD3 (Figure 1). The active VD3 hormone is a ligand derived from steroidal precursors in the skin via UVB-containing sunlight followed by

hydroxylations in the liver and kidney. Ligand-bound VDR-VD3 then heterodimerizes with its cognate co-receptor retinoid X receptor (RXR) to control expression of genes involved in calcium and phosphorus homeostasis, and bone mineral content.2 However, the scope of vitamin D and VDR biology has expanded to include a wide range of physiological cellular responses including profileration, differentiation, and immunomodulation. Due to the rich pharmacology of VDR, VD3 has been used clinically for the treatment of osteoporosis, psoriasis, cancer, and autoimmune diseases.3 However, its use is limited due to excessive absorption of mineral and pathological tissue hypermineralization. Over the last two decades, significant research has been directed at the identification of secosteroidal ligands that exhibit beneficial physiological function with improved therapeutic index relative to VD3.4 Unfortunately, most of these compounds functioned as full agonists and lack selective bone pharmacology. ED-71 (Figure 1), a seco steroid currently in phase II for osteoporosis, is one of the more promising therapeutics from these efforts, but it only exhibits a small improvement of safety relative to VD3.5 We and others have hypothesized that nonsecosteroidal ligands may allow for a larger separation between the desired bone formation effect and the undesired hypermineralization side effect by inducing a unique conformation of the receptor. For a given ligand this would, in principal, lead to specific VDR-ligand complex that is capable of recruiting cofactors that favor the transcription of genes specific for bone relative to those genes responsible for hypercalcemia.2 This type of tissue specificity has been demonstrated for other nuclear receptors such as the estrogen receptor.6 Thus, the identification of structurally divergent VDR ligands to explore this hypothesis became an important part of our strategy. One of the tactics we used to do so was to apply a fragment-based approach to find new chemical scaffolds for modulating VDR. Herein, we describe the discovery of new fragment ligands for VDR which were revealed by the use of a novel technique for fragment identification, hydrogen/deuterium exchange (HDX) mass spectrometry. Fragment-based drug discovery has proven successful for nuclear receptors within the same subfamily as VDR (e.g. the PPARs7), therefore we felt confident that it was a viable approach. Our strategy entailed the screening of a fragment library for binding to VDR, followed by biochemical determination of functional activity. Lastly, we planned to obtain protein co-crystals with selected hit scaffolds to determine the mode of binding and, ultimately, inform fragment growth. For the initial fragment screen, a targeted set of approximately10K compounds was assembled from the Lilly compound collection. As shown in Table 1, the cassette consisted mostly of fragment molecules as defined by the number of heavy atoms present, < 23. There were representative diversity block (1-3) as well as a VDR pharmacophore (VDR-like) and nuclear receptor (NR-like) blocks. Diversity 1-3 incorporated a range of physicochemical descriptors outlined in Table 1. Diversity 1, a “small fragment” cassette, contained 389 compounds with an average molecular weight (MW) of 117 with 6.6 heavy atoms (HA). Diversity blocks 2

and 3 were larger in number and higher in average MW 177-216 and HA12-14. The VDR-like block, contained fragments with structural motifs, such as carboxylic acids and alcohols, which are known to interact with VDR. Because ligands for nuclear receptors can be relatively large and lipophilic, we included a nuclear receptor block, (NR-like) containing compounds with known affinities to nuclear receptors related to VDR. . These compounds tended to have higher average MW (249) with HA’s (17). The fragments were tested in 384 well plates at 1 mM and 100 µM concentrations in a VDR binding fluorescence polarization assay. This resulted in 417 compounds with 50100% activity at 100 µM. False positives (approximately 200 fluorescent compounds) were removed resulting in 4.2% active rate. These binding hits were then tested in the RXR-VDR AlphaScreen® assay in both agonist and antagonist mode. In this confirmation sensing assay, DRIP205 was used as the coactivator which elicits a proximity-based signal when recruited to the VDR-RXR complex.8 When run in the presence or absence of an internal VDR agonist, this assay provides an antagonist or agonist response, respectively. When the hits from the binding assay were screened in agonist mode, none of the compounds showed any ability to recruit DRIP205 up to 500 µM. We postulate that the general lack of agonist activity results from insufficient interactions between the fragment and the ligand binding domain of the receptor to induce the required receptor dynamics in helices 10-12 (AF-2 surface) that allows for the recruitment of DRIP205.9 On the other hand, when screened in antagonist mode, i.e., in the presence of an internal non-secosteroidal VDR agonist,10 we were surprised to find that 34% of the compounds screened were active including 247 fragments that exhibited lean values of > 0.250. While antagonism of the VDR receptor is undesirable from a pharmacological standpoint, the interconversion between antagonism and agonism by structural modification of the ligand is well-documented for nuclear receptors.11 Thus, we thought that VDR antagonist fragments could serve as useful starting points for structural elaboration to tissue selective modulators that partially interact with helixes 10-12 of VDR LBD (vide infra). Of the 247 fragment hits, the majority were carboxylic acid derivatives. This structural motif is consistent with known VDR ligands which typically possess either a secondary alcohol, tertiary alcohol, diol, or benzoic acid moieties as polar anchoring groups (vida infra).4 In addition to carboxylic acids, other fragments with divergent structural features were identified that are novel in the VDR ligand space. Representative examples are shown in Table 2 and include a variety of diverse heterocycles containing functional groups with a wide range of pKa. Moreover, they represented starting points that are synthetically enabled to grow in multiple vectors. In order to expand structural diversity, the non-carboxylic acid fragments (lean > 0.25) such as 1-3, and 6 were used as a basis set to search for nearest neighbors within our internal compound collection. This hit expansion resulted in the identification of an additional set of > 2000 low molecular

weight compounds each with less than 20 heavy atoms. This diversity set was screened at high concentrations in the RXR/VDR assay and the resultant hits are shown in Table 3 which represent an additional source of fragment diversity. Compound 9 is the leanest fragment from the both the primary screen and actives assessment. Compounds 10 and 11 are structurally similar yet differ dramatically in the pKa of the hydroxy group. Fragment 12 contains a hexafluoroalcohol which is a known VDR pharmacophore.12,13 Compound 13 is one of 20 flavolins that were active in the RXR/VDR assay. With these chemical starting points (Tables 2 and 3) available for elaboration, we wanted to ascertain the binding mode of each within VDR in order to determine which would be the most fruitful to optimize. Traditionally, this would be done by cocrystallization of the ligand with the protein. However, in the case of VDR, attempts to do so were unsuccessful. In fact, there are relatively few protein crystal structures of small molecule ligands bound in VDR.14 As confirmation of the fragments in the ligand binding site was critical to our strategy, we began exploring orthogonal technologies to characterize fragment-protein interactions. Hydrogen/deuterium(HDX) exchange interfaced with mass spectrometry (HDX MS), has emerged as a rapid and sensitive approach for characterization of protein conformational dynamics altered by ligand binding.15 The HDX experiment measures the rate of exchange of protein backbone amide hydrogen with solvent deuterium in solution, the rate of which is related primarily to the hydrogen bonding environment of each amide. As such, any changes to the hydrogen bonding network of a protein following ligand binding can be detected with a differential HDX experiment (comparison of apo receptor to ligand bound receptor). These perturbations to HDX can be due to direct, as well as allosteric, interactions caused by protein interaction with small molecules, peptides, proteins and DNA.16 The HDX MS experiment has been shown by ourselves, and others, to be well suited to the characterization of nuclear receptors8,17,18,19,20,21 including VDR.22 During our previous HDX studies we have profiled how ligands with MW of 400-550 interact with nuclear receptors, however there are limited reports of the use of HDX to determine how fragment molecules, with smaller spatial volumes and fewer heavy atoms than traditional small molecules, interact with proteins.23 To this end, the ligands in Tables 2 and 3 were run in the HDX assay using a high ligand to protein ratio (200 to 1) in order to evaluate fragment interactions. Differential HDX analysis of the VDR LBD in the presence and absence of ligand were conducted. HDX kinetics of peptides derived from the VDR LBD were measured and the average difference in percentage of incorporated deuterium between apo VDR LBD and ligand-bound VDR LBD for the triplicate analysis of six on-exchange time points were determined and are reported in Table 4. A negative value represents an increase in protection to exchange (less dynamic) in that region of the LBD when bound to ligand as compared with apo,

whereas a positive value represents a decrease in protection to exchange (more dynamic). To our delight, of the 13 fragments examined by HDX, six elicited stabilization effects within the VDR LBD. To confirm whether the epitopes stabilized by the fragments were similar to those of the natural ligand (VD3), we compared the HDX profile for VD3 to that of the fragments. HDX data has been published for VD3,18 but contact points and perturbations are worth revisiting in order to understand the binding of the fragments. VD3 is a high affinity ligand which is revealed in its HDX fingerprint. This can be partially rationalized in the crystal structure due to key polar interactions.24 Deep in the pocket the cyclohexanediol A-ring interacts via H-bonds with TYR143 (helix 1), SER237 (helix 3), ARG274 (helix 5), and SER278 (helix 5) as shown in Figure 2. On the outer portion of the binding pocket facing the coactivator interface HIS305 (helix 6-7) and HIS397 (helix 11) make key contacts with the C-25 tertiary alcohol. The HDX data reveals dramatic stabilization (i.e. slowed HDX) at helixes containing the above polar residues as shown in Table 4. To aid in the visualization of HDX data we then map the data onto the VDR crystal structure (Fig 3). We then analyzed HDX data for fragment hits in order to determine extent of stabilization relative to apo VDR, as well as VD3, and also to determine a putative binding site in the VDR LBD. Analysis of the VDR LBD stabilizing fragments revealed that five out of six of the compounds stabilize helix 3, which contain key residue Ser237 (see above). This infers that these fragments contain functional groups that have polar interaction with Ser237 in a similar fashion as 3-OH of the cyclohexandiol of VD3. Three of the fragments also have impact on the stability of helix five suggesting polar interaction with Arg274, the other residue that has contact with 3-OH of VD3. From a structural perspective, one could conclude that fragments 6,8, and 12 contain rings that are at least partially mimicking the A-ring of VD3. Interestingly, all six fragments induce stabilization at helix7/8, a region remote of the LBD. The crystal structure of VD3/VDR does not show direct contact between the hormone and protein at this region. It should be noted that these helixes are located within the dimerization interface of the VDR/RXR complex and protection to exchange in this region is likely due to stabilization of a VDR homodimer. Out of all of the fragments run in the HDX assay, fragment 12 provided the most stabilization to deuterium exchange compared to apo VDR. In addition to stabilization in helices 3, 5, and 7/8 of up to 5%, 7%, and 7 % respectively, it exhibited slight stabilization at helix 3/4 region (3%) and helix 6 loop (6%). The helix 6 loop is of interest since it is involved in stabilization of the coactivator interface (outer helixes 10-12/AF2) and contains critical polar residue His 305. Close inspection of the molecular structure of 12 reveals a hexafluoroisopropanol group, a moiety found in known potent VDR agonists and typically replaces the C25-OH in D3.9-10 The CF3 groups inductively weaken the O-H bond strength thus favoring a stronger interaction

with His305. Figure 3 shows the HDX effects of 12 mapped on onto the crystal structure of VDR LBD-VD3. The color coded LBD found in the figure illustrates the coverage that 12 provides to VDR (Figure 2). In summary, a combination of VDR binding and biochemical screens, followed by structural analysis using MS detected hydrogen deuterium exchange of ligand bound VDR, enabled the identification of novel fragments for VDR that can serve as starting points for subsequent synthetic elaboration. The application of HDX to structurally enable fragment-based ligand identification for nuclear receptors is a unique approach and should prove useful for other targets, particularly those in which protein-ligand cocrystallization is challenging. Methods Quantitative alphascreen-based cofactor peptide interaction assays have previously been described for the analysis of VDR ligands.22 Differential HDX MS experiments were performed with a custom CTC twin HTS PAL liquid handling robot (LEAP Technologies, Carrboro, NC) interfaced with an Orbitrap mass spectrometer (Exactive, Thermo Fisher Scientific, Bremen, Germany) as described previously25.Exchange with D2O buffer was performed in triplicate for each condition (DMSO vs Fragment) at six different on-exchange time points (10, 30, 60, 300, 900 and 3600 s). Following onexchange, samples were quenched with 3 M urea containing 1% TFA at 1°C, digested on-line and analyzed with HPLC MS. Data processing were performed with custom software26 References and Notes 1. Evans, R.M. Science 1988, 240, 889. 2. Sutton, A.L.M.; MacDonald, P.N. Mol. Endocrinol. 2003, 17, 777. 3. Vieth, R. J. Bone. Miner Res 2007, 22, V64-V68. 4. Over 2000 secosteroids have been reported. For a review see Stayrook, K. R.; Carson, M. W.; Ma, Y. L.; Dodge, J. A. in Vitamin D; Feldman, D., Pike J. W., Adams, J.S. Eds; Elsevier, 2011; 3rd Ed, pp 1497-1508. 5. Sanford, M.; McCormack, P.L. Drugs 2011, 7(13), 1755. 6. Grese, T. A.; Sluka, J. P.; Bryant, H. U.; Cullinan, G. J.; Glasebrook, A. L.; Jones, C. D.; Matsumoto, K.; Palkowitz, A. D.; Sato, M.; Termine, J. D.; Winter, M. A.; Yang, N. N.; Dodge, J. A., Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14105 7. Artis, D.R.; Lin, J.J; Zhang, C.; Wang, W.; Mehra, U.; Perreault, M.; Erbe, D.; Krupka, H.I.; England, B.P.; Arnold, J.; Plotnikov, A.N.; Marimuthu, A.; Nguyen, H.; Will, S.; Signaevsky, M.; Kral, J.; Cantwell, J.; Settachatgull, C.; Yan, D.S.; Fong, D.; Oh, A.; Shi, S.; Womack, P.; Powell, B.; Habets, G.; West, B.L.; Zhang, K.Y.J.; Milburn, M.V.; Vlasuk, G.P.; Hirth, K.P.; Nolop, K.; Bollag, G.; Ibrahim, P.N.; Tobin, J.F. Proc. Natl. Acad. Sci. U.S.A., 2009, 106, 262.

8. Zhang, J.; Chalmers, M.J.; Stayrook, K.R.; Burris, L.L.; Garcia-Ordonez, R.D.; Pascal, B.D.; Burris, T.P.; Dodge, J.A.; Griffin, P.R. Structure 2010, 18(10), 1332 9. Carlberg, C.; Molnár, F. Curr. Top. Med. Chem. 2006, 6, 1243. 10.The fragments are referred to as antagonists because they displace a diarylmethane VDR agonist as evidenced by a dose response reversal of positive signal.. For examples of diarylmethanes see Bunel, E.E.; Gajewski, R.P.; Jones, C.D.; Lu, J.; Ma, T.; Nagpal, S.; Yee, Y.K. 2004 WO 2004048309. 11.Mortensen, D.S; Rodriguez, A.L.; Sun, J.; Katzenellenbogen, B.S.; Katzenellenbogen, J.A. ,Bioorg. Med. Chem. Lett., 2001, 11, 2521. 12.Chen, F.; Su, Q.; Torrent, M.; Wei, N.; Peekhaus, N.; McMasters, D.; Fisher, J.; Glantschinig, H.; Hodor, P.; Flores, O.; Reszka, A. Drug Dev Res 2007, 68, 51. 13.Perakyla, M.; Malinen, M.; Herzig, K.H.; Carlberg, C. Mol. Endocrinol 2005, 19, 2060. 14.Demizu, Y.; Takahashi, T.; Kaneko, F.; Sato, Y.; Okuda, H.; Ochiai, E.; Horie, K.; Takagi, K-I; Kakuda, S.; Takimoto-Kamimura, M. Bioorg. Med. Chem. Lett., 2011, 21, 6104. 15.Englander, S.W. J. Am. Soc. Mass Spectrom. 2006, 17, 1481. 16.Chalmers,M.J.; Busby,S.A.; Pascal,B.D.; West,G.M.; Griffin, P.R. Exp. Rev. Proteomics, 2011, 8, 43. 17.Bruning, J.B.; Chalmers, M.J.; Prasad, S.; Busby, S.A.; Kamenecka, T.M.; He, Y.; Nettles, K.W.; Griffin, P.R. Structure 2007, 15, 1258. 18.Hamuro, Y.; Coales, S.J.; Morrow, J.A.; Molnar, K.S.; Tuske, S.J.; Southern, M.R.; Griffin, P.R. Protein Sci. 2006, 15, 1883. 19.Dai, S.Y.; Burris, T.P.; Dodge, J.A.; Montrose-Rafizadeh, C.; Wang, Y.; Pascal, B.D.; Chalmers, M.J.; Griffin, P.R. Biochemistry 2009, 48, 9668. 20.Chalmers, M.J.; Wang, Y.; Novick, S.; Sato, M;; Bryant, H.U.; Montrose-Rafizdeh, C.; Griffin, P.R.; Dodge, J.A. ACS Med. Chem. Lett.2012, 3, 207. 21.Frego, L.; Davidson, W. Protein Sci. 2006, 15, 722. 22.Zhang, J.; Chalmers, M.J.; Stayrook, K.R.; Burris, L.L.; Garcia-Ordonez, R.D.; Pascal, B.D.; Burris, T.P.; Dodge, J.A.; Griffin, P. R. Structure 2010, 18(10), 1332. 23.Anand, G.S.; Krishnamurthy, S. WO 2012166056. 24.Rochel, N.; Wurtz, J.M.; Mitschler, A.; Klaholz, B.; Moras, D. Mol. Cell 2000, 5, 173. 25.Chalmers, M.J.; Busby, S.A.; Pascal, B.D.; He, Y.; Hendrickson C.L.; Marshall, A.G.; Griffin, P.R. Anal.Chem. 2006, 78, 1005. 26.Pascal, B.D.; Chalmers, M.J.; Busby, S.A. ;Griffin, P.R. J. Am. Soc. Mass Spectrom. 2009, 20, 601.

Figure 1. Chemical structures of seco-steroids of natural hormone 1,25 (OH)2-D3 (VD3) and ED-71.

OH

OH

H

H

HO

HO

OH

1,25 (OH)2-D3 (VD3)

OH O

OH

ED-71

Figure 2. Stabilization of VDR LBD by VD3 is mainly driven by key polar interactions between polar VD3 functional groups and VDR amino acids. (a) Average Differential HDX of VD3 mapped on VD3-VDR ligand crystal structure (PDB:1DB1), (b) VDR LBD polar amino acid and VD3 hydroxy interactions.

Figure 3. Average Differential HDX of fragment 12 mapped on VD3-VDR ligand crystal structure.

12

Table 1. Library #

Library Type

cpds

Avg MW

Avg # Heavy atoms

Avg rotatable bonds

Avg HB donors

Avg HB acc

Avg PSA

Avg clogP chem

Avg fraction sp3 C

Avg # rings

Avg aromatic density

1

Diverse small

389

117

6.6

0.17

0.9

2.2

37

0.1

0.50

1.0

0.4

2 3

Diverse Diverse

3290 3670

178 217

12.0 14.4

1.28 1.34

0.9 0.9

3.0 3.6

44 51

1.2 1.4

0.22 0.34

1.6 2.1

0.6 0.5

4 5

Acids NHRlike

1356 1568

196 249

13.4 17.2

1.79 2.54

1.4 0.8

3.9 2.7

63 40

1.3 3.2

0.17 0.21

1.7 2.3

0.6 0.6

Average Structural Properties of Fragment Libraries used in VDR Screen

Table 2 RXR/VDR activity and lean for VDR fragment hits from screening of libraries described in Table 1.

RXR/VDR IC50 (µM)a Leanb

Heavy atoms

1

1.01

0.535

11

2

5.37

0.439

12

3

14.2

0.404

12

4

2.77

0.370

15

5

16.7

0.299

16

6

6.98

0.344

15

7

20.0

0.294

16

Fragment

a

Structure

Using ant-Flag/streptavidin bead-based alphascreen technology, full-length human His-VDR and FlagRXR proteins were incubated with biotinylated DRIP205-2 33-mer cofactor peptides. A VDR agonist was added to form the VDR/RXR/DRIP205 complex. Fragments were added to this mixture with increasing concentration (10 doses from 0.025 to 500µM). The dissociation of the complex was quantified using a

Perkin Elmer Envision 2103 Multilabel Reader. Data analysis and curve-fitting were performed using GraphPad Prism software. bLEAN Ratio = -log(IC50) / Heavy-atom-count

Table 3 RXR/VDR activity and lean for fragment actives from nearest neighbor search of original hits in table 2.

Fragment Structure

RXR/VDR IC50, µM Leanb

8

440

0.280

12

9

6.83

0.517

10

43.2

0.336

13

67.1

0.321

13

1.30

0.280

21

0.194

0.305

22

10

Heavy atoms

OH O N

11

Cl

12 OH O

N

N O

13

HO

N

N

N

Table 4 Conformational Dynamics Induced by VD3 and VDR fragments Sequence SLRPKLSEEQQRIIA SLRPKLSEEQQRIIA ILLDAHHKTYDPTYSDF ILLDAHHKTYDPTYSDF LELSQL SMLPHLADL SMLPHLADL VSYSIQKVIGF FAKMIPGFRDLTSED FAKMIPGFRDLTSEDQ AKMIPGFRDLTSED AKMIPGFRDLTSEDQ LRSNESF RSNESFTMDDMS WTCGNQDYKYRVSDVTKAGHSLE VTKAGHSLE LIEPLIKF LIEPLIKF LIEPLIKFQVGLKKLNL LIEPLIKFQVGLKKLNLHEEE LIEPLIKFQVGLKKLNLHEEEHVLL QVGLKKLNL QVGLKKLNLHEEE ICIVSPDRPGVQDAAL IVSPDRPGVQDAAL IEAIQDRLSNTLQT AIQDRLSNTLQT YIRCRHPPPGSHLL YIRCRHPPPGSHLL YIRCRHPPPGSHLLY YIRCRHPPPGSHLLYAKM YIRCRHPPPGSHLLYAKM IQKLAD IQKLADL IQKLADLRSLNEEHSKQYRC IQKLADLRSLNEEHSKQYRC LRSLNEEHSKQYRC LRSLNEEHSKQYRC RSLNEEHSKQYRC RSLNEEHSKQYRC LSFQPECS SMKLTPLVL MKLTPLVL

NS

>-10

AA Position 119-133 (+2) 119-133 (+3) 134-150 (+2) 134-150 (+3) 219-224 (+1) 225-233 (+1) 225-233 (+2) 234-244 (+2) 244-258 (+3) 244-259 (+2) 245-258 (+3) 245-259 (+2) 273-279 (+2) 274-285 (+2) 286-308 (+3) 300-308 (+2) 309-316 (+1) 309-316 (+1) 309-325 (+3) 309-329 (+3) 309-333 (+3) 317-325 (+2) 317-329 (+2) 336-351 (+2) 338-351 (+2) 352-365 (+2) 354-365 (+2) 366-379 (+2) 366-379 (+3) 366-380 (+2) 366-383 (+3) 366-383 (+4) 384-389 (+2) 384-390 (+2) 384-403 (+3) 384-403 (+4) 390-403 (+3) 390-403 (+4) 391-403 (+2) 391-403 (+3) 404-411 (+1) 411-419 (+2) 412-419 (+2)

>-20

Structure Hinge/H1 Hinge/H1 H1 H1 Loop H3 H3 H3 H3/H4 H3/H4 H3/H4 H3/H4 H5 H5/S1 S2/H6/H7 Loop H7 H7 H7/H8 H7/H8 H7/H8 H7 H7/H8 H8/H9 H8/H9 H9 H9 H9/H10 H9/H10 H9/H10 H9/H10 H9/H10 H10 H10 H10/H11 H10/H11 H11 H11 H11 H11 LOOP H12 H12

>-30

Key residues

TYR143

SER237

ARG274 SER278 HIS305

HIS397

>-40

VD3 -2 -2 -17 -19 3 NaN -44 -48 -1 -1 1 0 -29 -33 NaN -22 -33 -36 -38 -27 -22 -39 -18 0 -2 -1 -3 -1 0 NaN 1 0 1 2 -18 -19 -26 -26 -25 -28 -4 NaN -28

>-50

3 -2 -2 0 0 0 NaN -3 -7 0 -1 -1 -1 0 0 1 0 -6 -7 -8 -5 -4 -8 -2 0 -1 0 -1 -1 0 NaN 0 -1 -1 -1 -2 -1 -1 -2 -1 -2 0 NaN -1

6 -2 -3 -1 -2 -1 -3 -3 -8 -1 -1 -1 -1 -5 -3 -2 -1 -8 -12 -9 -7 -4 -11 -6 -1 -1 0 -1 -2 -2 -1 -1 -1 -1 -1 -2 -3 -2 -3 -2 -3 -1 -1 -2

8 -2 -2 -1 -1 1 -2 -3 -9 0 -1 -1 0 -2 -2 -1 1 -8 -10 -11 -8 -6 -11 -7 0 -1 0 0 -1 0 1 -1 -1 -1 0 -3 -3 -3 -3 -3 -3 0 -1 -1

9 0 -1 0 0 -1 0 -1 -3 0 0 0 0 -2 -1 0 2 -4 -5 -7 -5 -4 -7 -4 0 0 0 0 0 0 0 0 0 0 0 -1 0 0 -1 -1 0 0 0 0

11 0 0 0 0 -2 -4 -4 -8 0 0 0 0 -3 -1 0 1 -5 -6 -7 -5 -3 -7 -4 0 0 0 0 0 0 -1 0 -1 -1 0 -1 -1 -1 -1 -1 -1 0 -2 -3

12 -2 -2 -1 -1 -5 -5 -4 -4 -2 -3 -1 -1 -7 -3 -2 -6 -5 -7 -7 -5 -4 -7 NaN -2 -2 0 0 -2 -2 -1 0 0 -2 0 -1 -2 NaN -3 NaN -1 -3 -3 -3

5 -4 -4 0 0 0 NaN -2 -2 0 0 0 0 0 0 0 0 -1 -2 -3 -2 -2 -3 -1 0 0 0 -1 0 0 NaN 0 0 1 0 0 0 -1 0 0 -1 0 NaN -1