Aurora_B_in_Complex_with_BI_847325
Crystal Structure of Aurora B in Complex with BI 847325 (PDB ID: 5EYK) from Xenopus laevis 
Created by: Monika Grabowska 

          The serine/threonine kinase Aurora B is a cell cycle regulatory protein present in all multicellular eukaryotic organisms (1). Aurora B is a component of the chromosomal passenger complex (CPC), which regulates key mitotic processes, ensuring correct chromosome alignment and segregation and proper execution of cytokinesis (2). Deregulation of the mitotic cell cycle is a key feature of oncogenesis, and cell cycle regulatory proteins such as Aurora B play a critical role in the pathogenesis and progression of cancer (3). Overexpression of Aurora B has been linked to aneuploidy and tumor formation. These features of Aurora B have made it a promising drug target for cancer therapies. In recent years, several Aurora B inhibitors, such as the ATP-mimetic BI 847325, have been studied as potential new treatments for cancer (4). The structure of Aurora B in complex with BI 847325 (PDB ID: 5EYK) from Xenopus laevis has elucidated the interaction of BI 847325 with Aurora B on a molecular level, providing important binding details that can be used to optimize drug design (4, 5, 6). 
          Aurora B is composed of Aurora B forms a heterotetrameric complex with inner centromere protein (INCENP). An INCENP subunit wraps around the N-terminal lobe of each of the Aurora B subunits, forming a molecular crown that helps stabilize Aurora B (8). The molecular weight of this complex is 79.2 kDa and its isoelectric point is 9.44 (7). The secondary structure of INCENP is 33% helical, consisting of three alpha helices: αA, αB, and αC (6, 8). 
          It is important to note that the structure of Aurora B in complex with BI 847325 does not cover the entire sequence of either Aurora B or INCENP. This structure contains only 276 residues of the 361-residue-long Aurora B and 58 residues of the 873-residue-long INCENP (6, 8). In discussing the structure of Aurora B in complex with BI 847325, residues are numbered according to their positions in the UniProt sequence. 
          The Aurora B:INCENP binding interface primarily involves hydrophobic interactions and hydrogen bonds. The Leu-807, Ala-810, and Ile-811 hydrophobic residues of the amphipathic αA helix of INCENP interact with the hydrophobic surface patch on Aurora B formed by Leu-109, Phe-117, and Phe-172 (8). Hydrogen bonds between the INCENP Gln-814 side chain and the alpha amino and alpha carbonyl groups of Ile-118 in Aurora B bring the C-terminal region of the INCENP αA helix into close contact with Aurora B (8). The INCENP αC helix interacts with Aurora B via its hydrophobic Pro-831, Leu-833, Leu-836, and Phe-837 residues (8). Specifically, the side chain of Phe-837 inserts deeply into the exposed hydrophobic pocket on Aurora B created by Ile-166, Leu-129, Glu-135, Leu-136, and Arg-139 (8). 
          INCENP plays a critically important role as an activator and regulator of Aurora B. The C-terminal IN-box region of INCENP provides the complementary hydrophobic motif needed to fill the hydrophobic pocket of the N-terminal lobe and thus activate the kinase (8, 9). The Pro-799, Trp-801, and Ala-802 residues of INCENP are the key mediators of this interaction. Due to its lack of a side chain, the Gly-96 residue lining the hydrophobic pocket enables the side chains of Trp-801 and Ala-802 to pack tightly into the pocket (8). The structure of Aurora B in complex with BI 847325 is incomplete, as it is missing a number of residues at the C-terminus of INCENP, including the residues that constitute the hydrophobic motif. However, this motif has been verified in another structure of the Aurora B:INCENP complex from the same organism (PDB ID: 2BFX). The binding of the INCENP hydrophobic motif residues to the hydrophobic pocket of Aurora B allosterically activates the kinase (9, 10).
          However, these interactions with INCENP are not sufficient to fully activate Aurora B. The activation of Aurora B by INCENP proceeds by a two-step process (8). INCENP binding induces Aurora B to autophosphorylate on the Thr-248 residue on its activation loop, resulting in an intermediate state of activation. Aurora B then phosphorylates the Thr-Ser-Ser motif within the IN-box of INCENP (corresponding to residues 848?850), which maximally activates its kinase activity. This mechanism enables Aurora B to regulate mitosis within cells. As the structure of Aurora B in complex with BI 847325 contains only residues 790-847 of INCENP, it represents an intermediately active state of Aurora B (6,8). 
          Aurora B has an ATP binding site at Lys-122 (6). When ATP binds to this site, the activated Aurora B is able to transfer a phosphate group from the ATP to one of its substrates, such as INCENP or histone H3, which is believed to play an important role in chromosome condensation (11). Thus, the Lys-122 residue enables activation of the downstream effectors of Aurora B. When this lysine residue is replaced with an arginine, phosphorylation of Thr-248 does not occur, indicating that this residue is critical to the catalytic activity of Aurora B (8). Another functionally important residue in Aurora B is Asp-216, which serves as the proton acceptor within the active site (6).  
          Proteins similar to the Aurora B:INCENP complex were identified using PSI-BLAST to analyze sequence similarity and the Dali server to analyze tertiary structure similarity. PSI-BLAST ranks comparison proteins according to their similarity measured by the E value, which depends on the similarity matrix and gap penalties (12). An E value less than 0.05 is usually regarded as significant (12). The Dali server compares intramolecular distances and computes a Z-score to measure similarity (13). A Z-score above 2 is considered significant (13).
          The complex of Aurora A with VX-680 and TPX2 from Homo sapiens (PDB ID: 3E5A) was one of the top matches returned by both PSI-BLAST and the Dali server, with an E value of 1e-128 and a Z-score of 37.7 (12, 13). The complex of Aurora B with BI 847325 and INCENP and the complex of Aurora A with VX-680 and TPX2 have 70% sequence identity, with a highly conserved catalytic domain but different N-terminal domains and different short C-terminal extensions (8, 12, 13). BI 847325 and VX-680 are both ATP-competitive Aurora kinase inhibitors, and thus perform identical roles in the two protein complexes (4, 14).
          Like Aurora B, Aurora A is a cell cycle regulatory protein involved in mitotic progression, but the two proteins have distinct functions and display distinct localization patterns during mitosis. Aurora A is predominantly involved in spindle formation, centrosome separation, mitotic entry via the G2-M transition, chromatid separation, and regulation of cytokinesis (15). Both Aurora B and Aurora A require the binding of a regulatory protein for activation. This regulatory protein is INCENP for Aurora B and TPX2 for Aurora A. Furthermore, both INCENP and TPX2 induce the autophosphorylation of a threonine residue in the activation loop of their associated kinases, corresponding to Thr-248 in Xenopus laevis Aurora B and Thr-288 in Homo sapiens Aurora A (8). Aurora A associates with the centrosomes during prophase, and TPX2 localizes activated Aurora A to the mitotic spindle during later stages of mitosis (15). In contrast, Aurora B is localized to the nucleus during prophase and then localizes to the inner centromere during metaphase. During later stages of mitosis, Aurora B relocates to the central spindle (15). 
          Although INCENP and TPX2 perform similar roles in Aurora B and Aurora A, respectively, the mechanisms of interaction between the kinases and their associated regulatory proteins differ substantially. Unlike the molecular crown formed by INCENP around Aurora B, TPX2 binds to Aurora A in two loosely connected discrete fragments, corresponding to TPX2 residues 7-21 and 30-43 (8). Residues 30-43 of TPX2 have no counterparts in INCENP, and the main chains of TPX2 and INCENP have opposite orientations (8).Three residues are implicated in ATP binding in Aurora A: Lys-143, Asp-256, and Asp-274 (6). In contrast, only one residue, Lys-122, is implicated in ATP binding in Aurora B (6). Furthermore, TPX2 activates Aurora A in one step, as compared to two for INCENP and Aurora B (8). However, both INCENP and TPX2 contain an "aromatic plug," which is formed by Phe-19 and Tyr-8 in TPX2 and Tyr-825 and Phe-837 in INCENP (8). In the aromatic plug, the aromatic residues project into a pocket on the surface of their associated kinase (8). 
          The differences in the function and localization of Aurora B and Aurora A despite their highly conserved catalytic domains can be attributed to a single amino acid difference: the presence of a glycine or an asparagine (16). In Homo sapiens, the Gly-198 residue in Aurora A, which corresponds to Gly-205 in Xenopus laevis, is replaced with the equivalent Aurora B residue Asn-142, corresponding to Asn-158 in Xenopus laevis (16). When the Gly-198 in Aurora A is mutated to an asparagine residue, Aurora A is able to bind INCENP but not TPX2 (17). This behavior may be explained in part by steric hindrance due to the asparagine side chain; the lack of a side chain at Gly-198 in Aurora A enables close interaction of TPX2 and Aurora A, whereas the bulkier side chain of Asn would impede close approach of TPX2 in this position (16). Thus this single residue difference mediates TPX2:Aurora A recognition, enabling TPX2 to distinguish between Aurora A and Aurora B (16). 
          One of the Aurora kinase inhibitors that has been investigated as a potential anticancer therapy is the compound BI 847325, which inhibits both Aurora A and Aurora B, as well as the mitogen-activated protein kinase kinase enzymes MEK1 and MEK2 (4). The indolinone scaffold of BI 847325 binds to the Lys-122 residue in Aurora B (4). BI 847325 competes with ATP for binding to this site; once bound, BI 847325 blocks the kinase activity of Aurora B, preventing Aurora B from phosphorylating any of its substrates and thus preventing cell division (4). BI 847325 was investigated in a phase I clinical trial in 2016, but further development of the drug was halted due to its insufficient inhibition of MEK (18). VX-680 is also being studied as a potential cancer treatment (14). Like BI 847325, VX-680 blocks Aurora kinase activity by binding to the ATP site (14).
          Aurora B and Aurora A play essential roles in cell cycle regulation; aberrant expression of these proteins has been implicated in oncogenesis. Aurora B and Aurora A are activated by the proteins INCENP and TPX2, respectively. A single residue in the catalytic domain, Gly-198 in Aurora A compared to Asn-142 in Aurora B, enables TPX2 to discriminate between Aurora A and Aurora B, resulting in differences in the function and subcellular localization of the proteins. The mitosis regulating functions of Aurora B and Aurora A make them promising anticancer drug targets. The ATP binding sites of Aurora B and Aurora A can be inhibited by small molecules such as BI 847325 and VX-680, providing possibilities for interference in cell mitosis which can be exploited to halt cell proliferation. The structure of Aurora B in complex with BI 847325 reveals the molecular-level details of BI 847325 binding to Aurora B:INCENP.