Protein kinases phosphorylate signaling proteins in cellular pathways to regulate essential biological activities. Primarily, they catalyze the transfer of a phosphate group from ATP to serine, threonine, or tyrosine residues of substrate proteins and induce a conformational change to activate the protein. The human genome consists of ~518 protein kinases, 1 making it one of the most represented protein families, with many of them involved in regulating cell division. 2 Thus, kinase inhibitors serve as the most sought out drug targets, mainly for cancer, evident from the ~37 FDA approved kinase inhibitors available in the market to‐date. 3 Among the kinase superfamily, the mitotic kinases belong to a class of kinases which can regulate the cell‐cycle entry, exit, checkpoint, and cytokinesis, deemed to be important for the growth and development of an organism. 4 To‐date, members of the cyclin‐dependent kinase family are the well‐studied mitotic regulators. 4 - 6 Members of the Polo, Aurora and NIMA mitotic kinases have also been studied for their involvement in cell cycle regulation. 4 - 6 A recent addition to this list is the human vaccinia‐related kinase 1 (VRK1) highlighted for its role in regulating cell‐cycle progression 7 and chromatin condensation. 8 , 9 VRKs are a family of Ser/Thr kinases, which belong to the casein kinase (CK1) group, consisting of three paralogous members: VRK1, VRK2, and VRK3. 10 VRK1 is mainly found in the nucleus, whereas VRK2 is found in ER membranes and the nuclear envelope. Although catalytic domain structures of VRK family reveal typical kinase fold, only VRK1 and 2 are catalytically active VRK's, 10 while VRK3 is catalytically inactive, which is attributable to its non‐canonical active site topology 11 (Supporting Information Fig. S1 ). In particular, the extra carboxyl‐terminal tail of VRK1 orients toward its catalytic site and forms multiple interactions that are critical for catalysis and structural stability. 12 Interestingly, its catalytic activity on histone H3 is oscillated during cell cycle and is regulated by macroH2A1, a histone variant. 13 VRK1 is discovered as an early response gene and is one of the important factors for the initiation of cell division process, 7 , 14 , 15 reflected on its overexpression in many cancerous tissues. 16 Recent studies have shown direct correlation of poor clinical outcomes of breast cancer patients was associated with high expression levels of VRK1 17 suggesting it as a promising cancer drug target. 16 - 19 VRK1 can phosphorylate itself (auto‐phosphorylate) as well as its interacting substrate proteins. During mitosis, VRK1 can phosphorylate (a) barrier‐to‐autointegration factor (BAF) to regulate the morphology of nuclear envelope 20 ; (b) H3's N‐terminal tail to control chromatin condensation 8 ; and (c) p53 which helps to stabilize DNA damage regulation. 21 Hence, dysregulation of VRK1 during mitosis can result in high cell proliferation. Recent studies led to the discovery of small molecule VRK1 inhibitors from natural products, such as luteolin, 22 brazilin, 23 and ursolic acid. 24 An extensive thermal shift screening using a well‐known kinase library, followed by crystal structure determination, revealed the first molecular basis of the VRK1 inhibitors, ASC24 (PDBID 3OP5), BI‐D1870 (PDBID 5UVF), and GW297361X (PDBID 5UKF), providing cues toward possible VRK1 selective inhibitor design. 25 While these inhibitors were shown to recognize the ATP binding pocket, their specificity as ATP‐competitive or ATP‐mimetic VRK1 inhibitors remains elusive. It has been shown that VRK1 shows poor selectivity toward known kinase inhibitors, 26 - 28 indicating that there is a scope for identifying VRK1‐specific inhibitors which could also have low promiscuity with other kinases. In this direction, we feel the lack of ATP‐bound VRK1 structure cripples the efforts toward identifying ATP‐competitive inhibitors “specific” to VRK1. The structural information delineating the underlying molecular interaction of VRK1 with ATP could facilitate the design of selective ligands for VRK1. To this end, we report here the NMR titration study to analyze the interaction of VRK1 with a non‐hydrolyzable ATP‐analog, AMP‐PNP, followed by the crystal structure of VRK1–AMP‐PNP complex highlighting its structural characteristics and comparison with other VRK1 structures.

Results and Discussion

NMR titration of VRK1 with AMP‐PNP The binding of AMP‐PNP on VRK1 was examined by performing a NMR study on a two‐dimensional 1H–15N TROSY spectroscopy (Fig. 1). The residues undergoing chemical shift perturbations upon AMP‐PNP titration were picked and analyzed, showing that the backbone amides of residues Gln45, Gly46, Gly49, Val 70, Lys71, Glu73, Phe81, Glu83, Leu84, Lys85, and Gln88 displayed chemical shift perturbations, while the residues Gly47, Cys50, and Val69 completely disappeared upon AMP‐PNP titration [Fig. 1(B) and (C)]. These perturbed residues are located on the highly flexible P‐loop, β3 strand, and αC helix. Residues Asp132, Arg133, and Phe134 of DRF motif, also known as the hinge region, along with the neighboring residues, Met131 and Gly135 also undergo chemical shift perturbations [Fig. 1(B) and (C)], indicating their roles in interacting with AMP‐PNP. Apart from these, residues His175, Gly176, Asp177, Ile178, Lys179, and Ser181 on the β6 strand (the catalytic loop region) and Asp197, Tyr198, and Gly199 of DYG motif, showed perturbations or disappearance upon AMP‐PNP binding [Fig. 1(B) and (C)]. The NMR titration studies apart from helping us to identify the perturbed residues on the canonical ATP binding pocket of VRK1 [Fig. 1(C)] also serves as a platform for fragment screening in future. Figure 1 Open in figure viewer PowerPoint The interaction between VRK1 and AMP‐PNP, an ATP analog characterized using NMR titration. (A) The overlay of 2D 1H–15N TROSY spectra of VRK1 in free state (red) with that of VRK1 in the presence of AMP‐PNP at a molar ratio of 1:5 (VRK1:AMP‐PNP; blue). A closer view of a few regions showing chemical shift perturbations or NMR signal disappearance upon the addition of AMP‐PNP are displayed as insets on the right. The direction of the shifts from free VRK1 (in red) toward AMP‐PNP bound form is indicated by black arrows and the residues are labeled for reference. (B) The chemical shift perturbation plot showing the regions of VRK1 affected upon the addition of AMP‐PNP, shown in the black box, and numbered according to the secondary structure elements of VRK1. The blue broken line represents the chemical shift perturbation (δΔ) threshold value set at 0.05. (C) The surface representation of VRK1 (PDB ID 2LAV), displaying residues 20 to 341 (in pale orange), with residues affected by the addition of AMP‐PNP labeled and colored in green.

Crystal structure of VRK1–AMP‐PNP To understand the atomic‐level interactions, the crystal structure of VRK1–AMP‐PNP complex was determined to a resolution of 2.07 Å. The crystals belonged to the orthorhombic space group, P2 1 2 1 2 1 , with four molecules in the asymmetric unit, with one AMP‐PNP along with a magnesium ion bound to each chain [Fig. 2(A)]. Overall, the VRK1–AMP‐PNP complex structure revealed an active site topology in ATP‐bound conformation of typical Ser/Thr kinases in which AMP‐PNP molecule docks its adenosine moiety into the active site pocket, while the phosphate group end orients toward the P‐loop. The AMP‐PNP is stabilized by a network of hydrogen bonds and non‐bonded interactions [Fig. 2(B) and Table 1], and the electron density map revealed that all its atoms are completely resolved [Fig. 2(C)]. Notable hydrogen bonds are those made by the adenosine nitrogen atoms with Asp132 and Phe134 backbone atoms (part of DRF hinge motif). One of the phosphate oxygen is hydrogen bonded to the Asp197 side chain (part of the DYG motif), while another phosphate oxygen forms a hydrogen bond with Lys71 side‐chain. In addition to this, the ribose sugar oxygen forms a hydrogen bond with Ser181 in Chain D, while not in others. Several non‐bonded interactions made by the adenosine and ribose rings with residues Ile51, Val69, Phe134, and Leu184, locks the adenosine end of AMP‐PNP within the active site. Residues from the DRF motif interact with adenine ring, while the DYG motif interacts with phosphate groups, bridged by the magnesium ion. AMP‐PNP adopts a similar orientation in the three chains (A, B, C) while there is a slight difference in that bound to the D‐chain. A closer look revealed the position of the bound magnesium is identical in the three chains, forming ionic interactions (~2.8 Å) with the neighboring atoms, while that bound to D‐chain is co‐ordinated (~2.1 Å) by the neighboring residues [Fig. 2(B) and Table 1]. In addition, a few water molecules stabilize the AMP‐PNP and magnesium by forming hydrogen bonded interactions [Fig. 2(B) and Table I]. It is worthwhile to mention that AMP‐PNP is a non‐hydrolyzable ATP analog expected to possess subtle changes to the mode of interactions made by ATP binding, nonetheless ATP analogs29 are being widely used for trapping the structural snapshot mimicking ATP binding. Figure 2 Open in figure viewer PowerPoint Crystal structure of VRK1–AMP‐PNP complex. (A) The cartoon representation of VRK1 bound to AMP‐PNP, in ball and stick mode, and a magnesium ion (Mg), shown as a green sphere. The major secondary structure elements are labeled for reference. (B) The interactions made by AMP‐PNP (shown in ball and stick), with the active residues of VRK1 (shown in stick mode). The hydrogen bonds and non‐polar interactions are shown as black and brown broken lines, respectively. The Mg ion (in green sphere) form a coordination geometry with AMP‐PNP phosphate groups and nearby Asp197 and Asn182 (shown as inset). (C) The 2Fo–Fc electron density map, contoured at 1σ cut‐off, showing density cover for all the atoms of AMP‐PNP and the co‐ordinated Mg ion. Table 1. Interactions made by AMP‐PNP (ANP) with VRK1. It is consolidated for all the four chains, while the magnesium coordination distances refer to Chain D only. For sake of comparison inhibitor (8E1) interactions with VRK1 (PDB ID 5UKF) are also provided Hydrogen bonds VRK1–ANP VRK1‐8E1 ANP Atom VRK1/ Mg2+ Atoms D–A distance (Å) 8E1 atom VRK1 atoms D–A distance (Å) ANP N6 Asp 132 O 2.8 8E1 N2 Asp 132 O 3.1 ANP O2A Lys 71 NZ 3.4 8E1 O Phe 134 N 2.9 ANP O2B Asp 197 OD1 3.0 8E1 N3 Asp 137 N 3.1 ANP N1 Phe 134 N 2.8 ANP O3’ Ser 181 O 2.9 ANP O1A Mg2+ 2.8 ANP O3A Mg2+ 3.2 ANP N7 H 2 O O 2.8 ANP O3’ H 2 O O 3.3 Mg coordination (in Chain D alone) Mg2+ ANP O1A 2.1 Mg2+ ANP O2B 2.0 Mg2+ Asp 197 OD1 2.3 Mg2+ Asn 182 OD1 2.1 Mg2+ H 2 O O 2.2 Non‐polar contacts VRK1‐ANP VRK1‐8E1 ANP Atom VRK1 Residues 8E1 Atom VRK1 Residues ANP C2 Phe134, Leu184 8E1 C Phe134, Gly135 ANP C4 Leu184 8E1 C1 Gly135 ANP C5 Leu184 8E1 C2 Gly135 ANP C6 Val69, Asp132, Phe134, Leu184 8E1 C5 Gly135 ANP C8 Ile51 8E1 C6 Leu184 ANP C5’ Ile51 8E1 C7 Leu184 ANP C2’ Leu184 8E1 C9 Val69, Phe134 ANP PA Ile51 8E1 C10 Met131, Phe134 ANP O1A Ile51, K71 8E1 C14 Phe48 ANP O2B Asp197 8E1 C15 Phe134 ANP O2’ Leu184 8E1 S Phe48, Ile51 ANP O4’ Ile51 8E1 O Val69, Arg133, Phe134 ANP O5’ Ile51 8E1 N1 Val196 ANP N1 Asp132, Arg133, Phe134 8E1 N2 Val69, Asp132, Arg133, Phe134 ANP N6 Val69, Met131, Asp132, Phe134 8E1 N3 Ser136, Asp137, Lys140

Structural comparison of VRK1–AMP‐PNP with apo and inhibitor counterparts The superposition of VRK1–AMP‐PNP complex on the apo structure12 (PDB ID 2LAV) and VRK1–inhibitor complex25 (PDB ID 5UKF) revealed a root‐mean‐square deviation (RMSD) of 1.70 Å (270 equivalent Cα atoms) and 0.56 Å (243 equivalent Cα atoms), respectively, indicating that the ligand bound structures are similar to each other compared to the apo form (Fig. S2). For the comparison studies, we used only the apo structure's representative model from its NMR ensemble. The formation of the salt‐bridge between Lys71 and Glu83, a primary feature of active kinases, was stronger in the presence of AMP‐PNP (at 2.8 Å) in comparison to the inhibitor bound form (3.7 Å), where the apo structure has a distance of 7.9 Å [Fig. 3(A)]. In addition, the distance between Glu83 and Tyr198 (of DYG) (7.2 Å) indicated that the α‐C helix adopts an “out‐like” conformation in the apo form, against the ligand bound conformation (a distance of 4.7 Å), which adopts an ‘in’ conformation.30 The conformation of the P‐loop from “Open” to “Close” was evident from the apo and inhibitor bound forms, respectively [Fig. 3(B)]. Although we could not trace electron density for the P‐loop residues in the AMP‐PNP bound form, the orientation of the residues on either side of this missing loop mimic that of the inhibitor bound conformation. We speculate that even in the AMP‐PNP bound form the P‐loop might adopt a “Close‐conformation” [Fig. 3(B)], though this needs further investigation. It was concluded that the P‐loop is a highly flexible region in all VRK1 crystal structures and could be traced in only one of chains in the inhibitor complex (Chain A of PDB ID 5UKF).25 Nonetheless, our NMR titration shows that the residues in the P‐loop (Gln45, Gly45, Gly46, and Gly49) are affected upon AMP‐PNP binding, validating their interaction. The DYG [Fig. 3(C)] and DRF [Fig. 3(D)] motifs are similar in the AMP‐PNP and inhibitor complexes compared with the apo form, in which a few side‐chains adopt different orientations. In all three structures, the “DYG‐in” orientation is observed. The orientation of the Met131, next to the DRF motif, also exhibits flexibility depending on the occupancy of the active site pocket. In the apo form and AMP‐PNP complex, Met131 protrudes toward the pocket, as opposed to the inhibitor bound complex, forming non‐bonded interactions with the ligand. It is evident that the inhibitor which docks deeper into the pocket compared to AMP‐PNP relocates the Met131 further away in comparison to the AMP‐PNP complex [Fig. 3(E)]. A similar flexibility was also observed when comparing the VRK2 apo and inhibitor complexes. It is to be mentioned that these active site Met residues are well‐conserved among the Ser/Thr kinases31 and more insights are required to explore their functional role in VRK1. Figure 3 Open in figure viewer PowerPoint Comparison of the VRK1–AMP‐PNP complex (green) with the apo (pale orange) and inhibitor‐bound (cyan) structures, highlighting the major signature motifs. (A) the salt bridge formed between the K71 and E83 (in stick mode) can be clearly visible in the AMP‐PNP (2.8 Å) and inhibitor (3.7 Å) bound structures, but not in the apo form (7.9 Å). (B) The P‐loop conformation adopts an open form in the apo structure, while is closed in inhibitor‐bound structure. As this region cannot be traced in the AMP‐PNP structure, it needs to be seen if it resembles a close‐conformation. The residues of the DYG (C) and DRF (D) motifs are also similar in the ligand‐bound conformations in comparison to the apo form. (E) Met131, shown in stick mode, can be observed to adopt a flexible orientation dependent on the nature of bound ligand, with the inhibitor pushing it the farthest from the active site pocket as this docks deeper. In (B) and (E), the ligands are shown in ball and stick mode for reference.