The chromosome passenger complex (CPC) is a complex of four proteins involved in the regulation of key mitotic events: correction of chromosome-microtubule attachment errors, assembly and stability of the spindle assembly checkpoint, and assembly and regulation of the contractile apparatus that drives cytokinesis (Carmena et al., 2012; Vader et al., 2006). The CPC is organized into two modules. The localization module consists of survivin, borealin, and the N-terminal part of INCENP, whereas the enzymatic module comprises Aurora B kinase and the C-terminal part of INCENP, called the IN-box. The enzymatic activity of Aurora B is essential for the regulatory functions of CPC (Kelly and Funabiki, 2009; Krenn and Musacchio, 2015; Meraldi et al., 2004), so Aurora B has been identified as an attractive target for cancer chemotherapy (Portella et al., 2011), with several inhibitors patented in the last decades (Jing and Chen, 2021).

The enzymatic activity of the [Aurora B/IN-box] is tightly regulated by autophosphorylation. The activity of the complex increases by at least two orders of magnitude after the complex phosphorylates itself (Zaytsev et al., 2016). There are two well-defined autophosphorylation sites in the [Aurora B/IN-box] enzyme complex. One is a threonine in the Aurora B activation loop, and the second is located at the C-terminus of the IN-box and consists of two consecutive serine residues in the TSS motif (Bishop and Schumacher, 2002; Honda et al., 2003; Sessa et al., 2005).

Until recently, the molecular understanding of Aurora B autoactivation was based on a model derived from the structure of the partially phosphorylated enzyme, in which the threonine in the Aurora B activation loop was phosphorylated but the TSS motif of INCENP was either absent (Sessa et al., 2005; Sessa and Villa, 2014) or unstructured (Elkins et al., 2012). Recently, a structural study of the [Aurora C/IN-box] complex, which is specific for meiosis but otherwise very similar to the [Aurora B/IN-box] complex, provided valuable insight into the conformation the fully phosphorylated enzyme adopts (Abdul Azeez et al., 2019). The crystal structure shows that the activation loop of Aurora C and the IN-box TSS motif interact in the phosphorylated state and stabilize the extended (active) conformation of the activation loop. Although the enzyme complex was crystallized with an inhibitor bound to the ATP-binding site, it is likely that the observed interaction between the IN-box and the activation loop represents a conformation corresponding to the active state of the enzyme complex. Crystal structures are extremely valuable as high-resolution snapshots of enzyme complex, but they lack the dynamic information about the enzyme complex that is required for a complete understanding of the autoactivation process. In addition, there is not crystal structure available of the [Aurora B/IN-box] complex in the unphosphorylated state and therefore, there is an important gap in understanding the conformational changes taking place during the activation process.

Proteins are dynamic macromolecules that exist in multiple conformations in their native state. An organized protein will explore a smaller number of conformations with less structural variability, than a disordered protein that will explore a larger number of conformations in the same time. In addition, there is an increasing evidence that the dynamic properties of an enzyme play a critical role in its catalytic functions (Ahuja et al., 2019; Tzeng and Kalodimos, 2012). For example, an increase in dynamics or local flexibility may facilitate the adoption of the different conformations required during the catalytic cycle (Boehr et al., 2006; Tzeng and Kalodimos, 2012; Xie et al., 2020). On the other hand, the transition from a highly dynamic, disordered enzyme to a more structured enzyme has also been documented as a form of regulation of enzyme catalysis (Wei et al., 2016; Xiao et al., 2015). In particular, an intensive line of research on protein kinases has shown that the internal dynamics associated with the movements of the enzyme’s subdomains regulate enzymatic activity (Ahuja et al., 2019; Pegram et al., 2021; Taylor and Kornev, 2011). While detailed dynamics analyses are available for several eukaryotic protein kinases activated by phosphorylation (Foda et al., 2015; Wang et al., 2019), including the closely related Aurora A (Cyphers et al., 2017; Dodson and Bayliss, 2012; Gilburt et al., 2017; Pitsawong et al., 2018; Ruff et al., 2018; Tomlinson et al., 2022), the effect of phosphorylation on the dynamic properties of the [Aurora B/IN-box] complex is not yet known.

Here, we use a combination of experimental (hydrogen-deuterium exchange coupled to mass-spectrometry [HDX-MS] and enzyme kinetics) and computational (molecular dynamics [MD]) approaches to follow the internal dynamics of the [Aurora B/IN-box] complex in solution - both in the unphosphorylated, inhibited, ([Aurora B/IN-box]^no-P) and fully phosphorylated, active, ([Aurora B/IN-box]^all-P) states. We found that in the absence of phosphorylation, there is structural disorder scattered along several regions of the enzyme complex. In contrast, in the fully phosphorylated enzyme complex, both Aurora B and the IN-box are structurally organized, resulting in a fully functional enzyme.

To investigate the contribution of individual phosphorylation sites to the dynamics and enzymatic activity, we used a protein ligation method to generate partially phosphorylated forms of the enzyme complex in which either only the activation loop of Aurora B ([Aurora B/IN-box]^loop-P) or only the TSS motif of the IN-box ([Aurora B/IN-box]^IN-P) is phosphorylated. Phosphorylation of each individual site in isolation has only a minor effect on enzymatic activity and structural stability, whereas simultaneous phosphorylation of the activation loop in Aurora B and the TSS motif in the IN-box synergistically affects the structure and dynamics of the enzyme complex, leading to a dramatic increase in kinase activity. Further, we identify phosphorylation in the activation loop of Aurora B as a rate-limiting intramolecular event in autoactivation. Finally, only when both sites are phosphorylated are the global movements of the N- and C-lobes of the enzyme (opening and closing of the active site and twisting between the lobes) coordinated with the movements of the activation loop, resulting in a fully functional kinase.

Our combined experimental and theoretical approach has allowed us to study in parallel unphosphorylated, partially phosphorylated, and fully phosphorylated [Aurora B/IN-box] and to provide the missing information necessary to build a more detailed model of [Aurora B/IN-box] autoactivation. Our model is important for understanding the role of protein dynamics in achieving full enzyme activity, and it may also serve as a starting point for the development of new Aurora B-specific inhibitors.

Using HDX and MD, we found that phosphorylation has a structuring effect on the [Aurora B/IN-box] complex, with the enzyme exploring fewer conformations and therefore becoming more rigid. Structuring after phosphorylation appears to be a general activation mechanism for the protein kinase family, and similar effects have been reported for protein kinase A and others (Hsu et al., 2008; Steichen et al., 2010).

A unique feature of Aurora B is its obligate association with the binding partner INCENP, which is also subject to phosphorylation. The recent crystal structure of [Aurora C/IN-box] showed that the phosphorylated IN-box interacts with the phosphorylated activation loop and stabilizes a more compact conformation, compatible with the active state of kinases. This structure implies a synergistic interaction between the two phosphorylation sites of the enzymatic complex (Abdul Azeez et al., 2019). We complement and extend these findings by using an experimental approach that is independent of the ability to crystallize the protein complex. Our approach addresses the lack of structural information for the unphosphorylated complex and the lack of information about structural dynamics and its possible role in [Aurora B/IN-box] autoactivation. Using a solution-based and computational approaches, we methodically studied the unphosphorylated, phosphorylated, and partially phosphorylated forms of the enzyme complex under the same conditions. Our enzyme kinetics experiments with the partially phosphorylated forms confirm that the two phosphorylation sites contribute synergistically to the Aurora B kinase activity. Moreover, the HDX experiments together with the MD simulations show a correlation between the dynamics of the two partners (Aurora B and the IN-box) and that phosphorylation of the activation loop in conjunction with phosphorylation in the IN-box is required to coordinate the dynamics between the N-terminal and C-terminal lobes of the kinase.

The HDX experiment clearly shows that the IN-box is a highly dynamic polypeptide chain, with most of its regions reaching maximal deuteration after only 3 min of D₂O incubation (Supplementary file 5), even in the phosphorylated state. Interestingly, the N-terminal part of the IN-box, which is the only part containing defined secondary structure elements, exhibits EX1-like HDX kinetics, suggesting that the IN-box^αA and IN-box^αB helices reversibly undergo folding and unfolding. Despite the reversible unfolding of the helices, the IN-box remains associated with Aurora B, which was confirmed by our mass photometry experiments (Figure 2 and Figure 1—figure supplement 4). The EX1 kinetics, and its relation to local unfolding of proteins under native conditions, has been described in detail in the literature. (Arrington and Robertson, 2000; Chitta et al., 2009; Ferraro et al., 2004; Krishna et al., 2004; Skinner et al., 2012; Sperry et al., 2012; Walters et al., 2013).

We observed EX1 kinetics in the N-terminal part of the IN-box independent of phosphorylation status, but the EX1 kinetics are faster in the unphosphorylated state, suggesting that the IN-box^805-825 fragment in [Aurora B/IN-box]^no-P switches more rapidly between the folded and unfolded states. This observation, together with the MD simulation analysis indicates that the higher unfolding rate in the IN-box N-terminal region of the unphosphorylated state transfers disorder to the adjacent N-lobe of Aurora B and in this way negatively affects kinase activity (Figure 2D and F). In summary, we provide strong evidence that in the unphosphorylated form of the enzyme complex, the IN-box acts as a negative regulator.

On the other hand, phosphorylation of the Aurora B activation loop leads to partial structuring within Aurora B and to a substantial conformational change of the IN-box, as well as to the adoption of an enzyme conformation closer to that required for efficient catalysis. However, the absence of phosphate groups on the IN-box continues to interfere with the optimal arrangement of the enzyme complex. Finally, when both Aurora B and the IN-box are phosphorylated, the enzyme complex is structured and coordinated in its global movements (Figure 7). Thus, in the fully phosphorylated form of the enzyme complex, the phosphorylated IN-box acts as a positive regulator that stabilizes the productive enzyme conformation.

Figure 7 with 1 supplement see all

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The results of the MD simulations are consistent with the HDX analysis and highlight the entropic nature of the IN-box. However, the C-terminal region of the IN-box adopts a different conformation in our [Aurora B/IN-box]^all-P simulation than in the [Aurora C/IN-box]^all-P crystal structure, raising the possibility that the observed [Aurora B/IN-box]^all-P structure is either an intermediate on the activation coordinate or an alternative conformation in the fully phosphorylated state. To better understand the role of phosphorylation on the IN-box conformation, we performed simulations on [Aurora C/IN-box] in the fully phosphorylated state, [Aurora C/IN-box]^all-P, and in the partially phosphorylated state, [Aurora C/IN-box]^loop-P, using the [Aurora C/IN-box] crystal structure as a starting model (Figure 7—figure supplement 1). These simulations confirmed that the residues following the TSS motif are highly flexible, even in the fully phosphorylated state. In addition, MD simulations on an [Aurora C/IN-box]^loop-P show that the absence of the IN-box phosphates in the [Aurora C/IN-box] structure results in relaxation of the longer C-terminal segment of the IN-box, which contains the TSS motif. In this simulation, the IN-box is less organized, overall (higher RMSD), than in the [Aurora C/IN-box]^all-P simulation and the IN-box^886-903 rapidly loses association with the phosphorylated activation loop and moves toward the C-lobe of the kinase to adopt a similar, but not identical, conformation to that observed in our original simulation, [Aurora B/IN-box]^loop-P (Figure 7—figure supplement 1, inset). The observed behavior confirms that the C-terminal part of the IN-box is a highly entropic unit that follows fast diffusion dynamics directly controlled by the phosphorylation state of the system, with the highest degree of disorder in the unphosphorylated state, partially organized in the [Aurora B(C)/IN-box]^loop-P state, and better organized, although still highly dynamic, in the [Aurora B(C)/IN-box]^all-P.

The high dynamics of the IN-box is most likely the main reason for the difficulties in obtaining the crystal structure of [Aurora B/IN-box]^all-P, as reported by Sessa et al., 2005. Therefore, according to our analysis, it is likely that there are few equally stable and productive conformations of [Aurora B/IN-box]^all-P. [Aurora C/IN-box]^all-P more likely represents the more stable conformation of the active state while the conformation observed in the [Aurora B/IN-box]^all-P simulation, described here, is probably a productive intermediate along the activation coordinate. Limitations in the simulation time and the very entropic nature of the IN-box C-terminal region may be the reasons our simulation did not reach the IN-box conformation observed in the [Aurora C/IN-box]^all-P crystal structure.

Overall, the IN-box plays a major role in the structural conformations adopted by Aurora B and in the synchronization of Aurora B subdomain motions observed in the active state of the enzyme complex. The domain movements and correlated motions within kinase lobes have been proposed to be part of the enzymatic turnover (Kumar et al., 2018; Kuzmanic et al., 2017; Masterson et al., 2010; Xiao et al., 2014). The strongest evidence for the coupling of global dynamics of protein kinase lobes with catalysis comes from NMR experiments on protein kinase A, where the opening and closing of the active site correlates very well with the catalytic turnover of the enzyme (Masterson et al., 2011). Since only the fully active [Aurora B/IN-box]^all-P exhibits synchronized global motions, we propose that global domain movement is directly linked to the catalytic cycle of this enzyme complex and likely controls substrate binding and product release.

In the present study, we use the [Aurora B/IN-box] complex containing only the INCENP fragment required for interaction with Aurora B, the IN-box, whereas the full-length INCENP has a long N-terminal part, which raises the question of whether or not the dynamics of the IN-box are different in the context of the full-length INCENP. The IN-box was previously defined as the minimal region of INCENP that binds Aurora B (Sessa et al., 2005), and phosphorylation of [Aurora B/IN-box] results in similar enzymatic activation as [Aurora B/INCENP^full-length] (Wang et al., 2011a; Zaytsev et al., 2016), suggesting that the IN-box is the only region of INCENP that is important and necessary for the regulation of kinase activity by phosphorylation. This is a strong indication that the dynamic changes observed in the IN-box upon phosphorylation of [Aurora B/IN-box] most likely follow the same trend in the context of full-length INCENP. At the same time, it is noteworthy that the N-terminal region of INCENP is required for the localization of the CPC at the inner centromere, resulting in a high local concentration of the [Aurora B/INCENP] complex, which is crucial for the rapid intermolecular activation of the kinase complex (Wang et al., 2011a; Zaytsev et al., 2016).

It has been previously proposed that autoactivation of [Aurora B/IN-box] occurs via a two-step mechanism with a slow cis-step and a fast trans-step (Zaytsev et al., 2016). Following our kinetic and MD analysis with partially phosphorylated enzyme complexes, we can now confirm this model and conclude that phosphorylation of the Aurora B activation loop is the slow intramolecular process followed by rapid trans phosphorylation of the IN-box TSS motif. It is important to emphasize that in the proposed model, the slow intramolecular step is obligatory only when the entire population of [Aurora B/IN-box] is dephosphorylated. The initial intramolecular step is followed by intermolecular propagation of activation, which may also include the activation segment.

Phosphorylation gradients of Aurora B substrates in the cell have been identified (Fuller et al., 2008; Wang et al., 2011a), and these long-range gradients play a regulatory role in mitosis (Afonso et al., 2014; Ferreira et al., 2013; Uehara et al., 2013). In addition, a strong evidence for the existence of a steep gradient of Aurora B activity at metaphase chromosomes has been reported and it has been suggested that the kinetics of Aurora B activity plays an important role in the formation of this gradient (Liu et al., 2009; Zaytsev et al., 2016). Here, we found that the partially phosphorylated [Aurora B/IN-box] intermediates have very low kinase activity (5–10%) which, together with the two-step Aurora B autoactivation, is consistent with the ability to form a steep phosphorylation gradient. Thus, a small increase in phosphatase concentration in a local cell region such as the kinetochore could cause a sharp decrease in kinase activity in the kinetochore region, whereas in a nearby region, Aurora B kinase activity could remain high.

The very similar Aurora A (72% identity), which associates with a different binding partner, TPX2, can be activated to an appreciable extent independently, either only by association with TPX2 or only by phosphorylation in the activation loop (Dodson and Bayliss, 2012). Two pathways of Aurora A activation are used separately at the physiological level. The pool of Aurora A involved in chromatin-driven microtubule nucleation is regulated exclusively by association with TPX2 (Kufer et al., 2002) and phosphorylation in the activation loop is not observed (Levinson, 2018). On the other hand, Aurora A activation at the centrosome depends only on phosphorylation of the activation loop and does not involve TPX2 binding (Joukov et al., 2010). While Aurora A has evolved to allow two independent activation pathways, activation of Aurora B, which is always in complex with IN-box, follows a single synergistic activation pathway that requires the coincidence of two events: phosphorylation of threonine in the activation loop and phosphorylation of the binding partner (TSS motif in the IN-box).

The present study demonstrates the important role that dynamics plays in regulating the enzymatic activity of [Aurora B/IN-box]. Our study explains and highlights the importance of IN-box (specific and obligatory Aurora B partner) as a necessary structural and strong regulatory element of the enzyme complex. The new findings suggest that the interface between Aurora B and the IN-box represents a target area for the development of a new type of Aurora B-specific drugs that would disrupt the allosteric changes required for activation. Our results contribute to a better understanding of the biophysical basis for the switch-like transition in Aurora B catalytic activity upon autophosphorylation (Zaytsev et al., 2016), a feature that underlies the intracellular spatial pattern of Aurora B substrate phosphorylation essential for proper chromosome segregation and cell division.

All data generated or analysed during this study are included in the manuscript and supporting files. The mass spectrometry proteomics data are available through the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD038935. Simulation data are freely available for download at the NIRD repository through the link: https://doi.org/10.11582/2023.00028.

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Author details

1. Dario Segura-Peña

   Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Faculty of Medicine, Oslo, Norway

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   dario.segura-pena@ncmm.uio.no

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6695-1042

2. Oda Hovet

   1. Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Faculty of Medicine, Oslo, Norway
   2. Department of Biosciences, University of Oslo, Oslo, Norway
   3. Hylleraas Centre for Quantum Molecular Sciences, University of Oslo, Oslo, Norway

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Hemanga Gogoi

   Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Faculty of Medicine, Oslo, Norway

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Jennine Dawicki-McKenna

   Department of Biochemistry and Biophysics, Penn Center for Genome Integrity, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Data curation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Stine Malene Hansen Wøien

   Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Faculty of Medicine, Oslo, Norway

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Manuel Carrer

   1. Hylleraas Centre for Quantum Molecular Sciences, University of Oslo, Oslo, Norway
   2. Department of Chemistry, University of Oslo, Oslo, Norway

   Contribution

   Visualization

   Competing interests

   No competing interests declared

7. Ben E Black

   Department of Biochemistry and Biophysics, Penn Center for Genome Integrity, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

8. Michele Cascella

   1. Hylleraas Centre for Quantum Molecular Sciences, University of Oslo, Oslo, Norway
   2. Department of Chemistry, University of Oslo, Oslo, Norway

   Contribution

   Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   For correspondence

   michele.cascella@kjemi.uio.no

   Competing interests

   No competing interests declared

9. Nikolina Sekulic

   Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, Faculty of Medicine, Oslo, Norway

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   nikolina.sekulic@ncmm.uio.no

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8027-9114

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