Redox priming promotes Aurora A activation during mitosis

Daniel C. Lim

1,*

Vladimir Joukov

T. Justin Rettenmaier

3,4

Akiko Kumagai

William G.

Dunphy

James A. Wells

Michael B. Yaffe

1,6,*

MIT Center for Precision Cancer Medicine, Koch Institute for Integrative Cancer Research,

and Departments of Biological Engineering and Biology, Massachusetts Institute of Technology,

Cambridge 02139, MA, USA

N. N. Petrov National Medical Research Center of Oncology, Saint Petersburg 197758, Russian

Federation

Jnana Therapeutics, Boston 02210, MA, USA

Departments of Pharmaceutical Chemistry & Cellular and Molecular Pharmacology, University of

California San Francisco, San Francisco 94158, CA, USA

The Division of Biology and Biological Engineering, California Institute of Technology, Pasadena,

CA 91125, USA

Divisions of Acute Care Surgery, Trauma, and Surgical Critical Care, and Surgical Oncology,

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

Abstract

Cell cycle-dependent redox changes can mediate transient covalent modifications of cysteine

thiols to modulate the activities of regulatory kinases and phosphatases. Our previously reported

finding that protein cysteine oxidation is increased during mitosis relative to other cell cycle

phases suggests that redox modifications could play prominent roles in regulating mitotic

processes. The Aurora family of kinases and their downstream targets are key components of

the cellular machinery that ensures the proper execution of mitosis and the accurate segregation

of chromosomes to daughter cells. In this study, X-ray crystal structures of the Aurora A

kinase domain delineate redox-sensitive cysteine residues that, upon covalent modification, can

allosterically regulate kinase activity and oligomerization state. We showed in both

Xenopus laevis

egg extracts and mammalian cells that a conserved cysteine residue within the Aurora A activation

loop is crucial for Aurora A activation by autophosphorylation. We further showed that covalent

disulfide adducts of this residue promote autophosphorylation of the Aurora A kinase domain.

These findings reveal a potential mechanistic link between Aurora A activation and changes in the

intracellular redox state during mitosis, as well as provide insights into how novel small molecule

inhibitors may be developed to target specific subpopulations of Aurora A.

Corresponding author. danlim@mit.edu (DCL); myaffe@mit.edu (MBY).

Author contributions

: D.C.L., M.B.Y., V.J., T.J.R., and J.A.W. designed the experiments. D.C.L., V.J., A.K. and T.J.R. performed the

experiments. D.C.L., M.B.Y., V.J., T.J.R., J.A.W. and W.G.D. performed data processing, analysis and interpretation.

Competing interests

: The authors declare that they have no competing interests.

HHS Public Access

Author manuscript

Sci Signal

. Author manuscript; available in PMC 2021 October 13.

Published in final edited form as:

Sci Signal

. ; 13(641): . doi:10.1126/scisignal.abb6707.

Author Manuscript

INTRODUCTION

The Aurora kinases are key mitotic kinases conserved in all eukaryotes (

). Three Aurora

kinase paralogs are expressed in mammals and share a highly conserved C-terminal

Ser/Thr protein kinase domain. Aurora A plays a critical role in regulating centrosome

maturation, mitotic entry, and spindle organization as well as correcting improper

kinetochore-microtubule attachments near spindle poles (

–

), while Aurora B plays a

similar role in destabilizing improper kinetochore-microtubule attachments at the metaphase

plate (

). Aurora A is diffusely distributed at low levels in the cytoplasm during interphase

but becomes concentrated at centrosomes and spindle microtubules proximal to the spindle

poles during late G2 and M phases (

). Aurora B localizes to centromeres in early mitosis

and subsequently to the central spindle during anaphase and to the cleavage furrow and

midbody during cytokinesis (

). Aurora C shares similar functions and localizations as

Aurora B, but its expression is normally restricted to germ cells and is required for

spermatogenesis and oocyte development (

Because Aurora A and B are expressed in all mitotically active cells and are increased

in abundance during late G2 and M phases (

), it is not surprising that the Aurora

kinases are overexpressed in multiple cancer types (

). In particular, the gene encoding

Aurora A, located within chromosome 20q13, is frequently amplified in breast, colorectal

and bladder tumors, and also in ovarian, prostate, neuroblastoma and cervical cancer cell

lines (

). Aurora A overexpression is associated with genomic instability and is a poor

prognostic marker in patients with head and neck squamous cell carcinomas, ovarian

and gastrointestinal tumors, colorectal cancer liver metastases, glioblastomas, and breast

carcinomas. These findings have led to intense pharmacological interest in developing small

molecule inhibitors of Aurora A. Despite promising results seen in pre-clinical models,

none of the ATP-competitive Aurora A inhibitors available to date have shown efficacy

in the treatment of cancer patients (

). Whether covalent or allosteric inhibitors

might function as better inhibitors is not known, but intriguingly, several type II Aurora

A inhibitors that specifically induce an inactive conformation of the kinase domain have

been shown to increase survival in a mouse model of N-Myc-driven neuroblastoma by

disrupting the ability of Aurora A to bind to and stabilize N-Myc (

) independent of

its catalytic activity. Clearly, a better understanding of the mechanisms that control Aurora

A kinase localization, activity, structural conformation and interactions with other proteins

could lead to more effective development and application of Aurora A inhibitors, both as

cancer therapeutics and as research tools.

The regulation of Aurora A activity and function is complex and depends on both the

subcellular localization of different pools of Aurora A, and the presence of specific binding

partners (

). The best characterized among these interactors are Bora, CEP192 and

Targeting protein for Xklp2 (TPX2), which bind directly to Aurora A in a mutually exclusive

manner (

). TPX2 localizes Aurora A to spindle microtubules (

), and its binding

allosterically activates the Aurora A kinase domain by stabilizing an active conformation

of the activation segment (

). Bora binds to both Aurora A and Polo-like kinase

1 (Plk1) specifically in the cytoplasm, followed by Plk1 activation by promoting Aurora

A-mediated phosphorylation of Thr

210

within the Plk1 activation loop (

). Plk1, in

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turn, then promotes activation of the cyclin-dependent kinase 1 (Cdk1)/cyclin B complex to

trigger mitotic entry (

–

An alternative mechanism is involved in regulating Aurora A and Plk1 activity at

centrosomes (

). There, CEP192 recruits both Aurora A and Plk1 to centrosomes

where Aurora A undergoes activation through autophosphorylation of Thr

288

within

its activation loop, through a poorly understood process. The activated Aurora A

subsequently phosphorylates and activates centrosomal Plk1. This centrosomal Aurora

A-Plk1 phosphorylation cascade is required for centrosome maturation and microtubule

nucleation, as well as for centrosome separation and bipolar spindle formation (

Although

Xenopus

CEP192 (xCEP192) is required for recruitment of

Xenopus

Aurora

A (xAurora A) to centrosomes, experiments in the

Xenopus

egg extract system have

shown that xAurora A activation/autophosphorylation can also be induced in the absence

of centrosomes by forced dimerization through addition of a bivalent antibody to xAurora

A (anti-xAurora A) to the egg extracts (

). However, no xAurora A autophosphorylation is

observed if the extracts are first depleted of xCEP192 prior to addition of the anti-xAurora A

dimerizing antibody. This suggests that in addition to recruiting xAurora A to centrosomes

and facilitating dimerization, other factors are required for xAurora A activation. We believe

this activation process involves redox modifications of Aurora A itself.

We have previously reported that overall levels of protein thiol oxidation increase as

mammalian cells progress through the cell cycle (

). Elegant studies by Rhee, Finkel,

Carroll and their colleagues, and others have shown that modification of cysteine residues by

thiol oxidation can directly regulate the activity of proteins, including cell cycle regulatory

kinases and phosphatases (

–

). Furthermore antioxidant and redox sensor proteins such

as peroxiredoxins also contain highly reactive cysteine residues (

). Reactive cysteine

residues in all of these proteins can be reversibly oxidized to sulfenic and sulfinic acids (

), or further oxidized irreversibly to sulfonic acid. Cysteine sulfenic acids can further react

with other thiols, including other protein cysteine residues and thiol-containing metabolites,

to form a range of thiol-disulfide species that in turn can undergo further thiol-disulfide

exchange reactions. Through these exchanges, redox signals leading to disulfide bond

formation can be transduced directly onto signaling molecules and from sensor proteins to a

wide range of target proteins. Links between redox modifications and cell cycle regulation

have previously been reported, including the activation of growth factor signaling pathways

via the oxidative modifications of cysteine residues that stimulate receptor tyrosine kinases

and inhibit protein tyrosine phosphatases (

). Reversible oxidative inhibition of

Cdc14B phosphatase to promote CDK1 signaling during mitosis, for example, has also

been reported (

). Along these lines, we previously reported that overall protein thiol

oxidation is increased during mitosis relative to other cell cycle phases (

), which likely

reflects the existence of additional redox-dependent regulatory mechanisms with important

roles in mitosis. In a search for new inhibitors of Aurora A, we identified several redox

sensitive cysteine residues within the Aurora A kinase domain. Our data indicate that Cys

290

within the activation loop is essential for Aurora A autophosphorylation at Thr

288

, and that

disulfide modifications of Cys

290

prime the kinase domain for autophosphorylation upon

dimerization.

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RESULTS

Conserved redox-sensitive cysteines within the Aurora A kinase domain can be covalently

modified to induce an inactive homodimeric conformation

In an effort to identify novel small molecule inhibitors of the Aurora A kinase domain, we

created a chimeric construct consisting of a fragment of TPX2 (residues 7 to 20) fused to the

N-terminus of the Aurora A kinase domain (residues 116 to 389), hereafter referred to as t

Aurora A. Like other AGC family kinases, the Aurora A kinase domain contains a conserved

hydrophobic patch adjacent to helix C, commonly referred to as the PDK1-interacting

fragment (PIF) pocket. A C-terminal hydrophobic motif in most AGC kinases docks

in cis

on to the PIF pocket to stabilize an active conformation of the kinase domain. Although

Aurora A does not contain a hydrophobic motif, Tyr

and Tyr

within TPX2 functionally

serve this purpose

in trans

(

). Our t-Aurora A construct thus contains a hydrophobic

motif

in cis

, which likely accounts for the improved overall stability relative to constructs

of the Aurora A kinase domain alone and allowed us to obtain more ordered crystals

with improved diffraction quality. Similar to other Aurora A kinase domain constructs,

t-Aurora expressed in bacteria in the absence of phosphatase co-expression or treatment is

phosphorylated on both Thr

287

and Thr

288

within the activation loop. The structure of this

autophosphorylated construct was then determined under a variety of chemical modification

and buffer conditions, revealing unexpected conformational differences in the kinase domain

depending on the modification state of key Cys residues. As expected, the structure of fully

reduced and autophosphorylated t-Aurora A showed a monomeric kinase domain adopting

the canonical protein kinase fold with an N-terminal lobe mainly consisting of a

-sheet and

two

-helices (

B and

C), and a larger predominantly

-helical C-terminal lobe (Fig. 1A).

This structure closely resembles the previously determined structure of the Aurora A kinase

domain in complex with the N-terminal 43 residues of TPX2 (PDB code 1OL5) (

), with

the two structures sharing an rms deviation of 0.64 Å over 264 C

atoms and show an active

conformation with a fully ordered activation segment phosphorylated on Thr

288

and Thr

287

However, a crystal structure of t-Aurora A obtained with cacodylate as the pH buffering

agent, unexpectedly revealed covalent modification of Cys

247

and Cys

290

with dimethyl

arsenic adducts (

), inducing a conformational rearrangement of the kinase domain (Fig.

1A, shown in blue). Furthermore, the structure of this cacodylate-modified form of t-Aurora

A showed that the kinase domain dimerized via a displaced activation segment that is

swapped between two symmetry-related molecules within the crystal structure (Fig. 1B),

with the dimerized kinase domains oriented with their N-terminal lobes pointing in roughly

orthogonal directions.

It is well known that the conformation of the activation segment within the C-terminal lobe

is a key determinant of whether a kinase domain is in a catalytically active or inactive

state (

). In particular, the conserved DFG motif at the N-terminal end of the activation

segment is observed in multiple conformations in kinase domain crystal structures, with

the DFG-in conformation being required for kinase activity. In the DFG-in conformation,

the phenylalanine side chain within the DFG motif points inward and is stacked within a

conserved column of buried hydrophobic side chains referred to as the regulatory spine (

). Notably, the activation segment in our t-Aurora A structure with cacodylate-modified

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cysteine residues, is in an inactive DFG-out conformation, in which the side chain of Phe

275

within the DFG motif is flipped outward into the ATP binding pocket (Fig. 2A), resulting in

the misplacement of key catalytic residues, including the Mg

-coordinating Asp

274

within

the DFG motif, and adenylyl-imidodiphosphate (AMP-PNP, a non-hydrolysable analogue

of ATP) bound in a distorted non-catalytic conformation. Superposition of the DFG-out

cacodylate-modified form with the fully reduced and active DFG-in t-Aurora A structure

shows a steric clash between the dimethyl-arsenic adduct on Cys

247

with the side chain of

Phe

275

in the DFG-in conformation, indicating that covalent modification of the Cys

247

side

chain would allosterically inhibit Aurora A kinase activity. In addition, an overall widening

of the active site cleft and displacement of the side chain of Glu

181

on helix

C results

in an opening of a small hydrophobic cavity adjacent to the ATP-binding pocket in the

cacodylate-modified structure, where it is occupied by an oxidized DTT molecule. This

cavity is also observed in the structure of an Aurora A kinase domain bound to a specific

5-aminopyrimidinyl quinazoline inhibitor (PDB code 2C6E), where it is occupied by the

benzamide moiety of this type II kinase inhibitor (

) (Fig. 2B). These observations suggest

that compounds that selectively modify Cys

247

may provide a scaffold for the development

of novel covalent non-ATP-competitive Aurora A inhibitors, and further suggests that such

inhibitors which re-structure the active site may facilitate the binding of, and thereby work

in conjunction with, existing Aurora A inhibitors like the 5-aminopyrimidinyl quinazoline

compounds.

A tethering screen of Cys

247

-directed disulfide compounds identifies the activation loop

cysteine as a site of redox modification that promotes Aurora A autophosphorylation

To identify Cys

247

-modifying Aurora A inhibitors, we next conducted a high throughput

mass spectrometry-based tethering screen (

) of 880 disulfide containing molecules to

identify compounds that can stably label Cys

247

through thiol-disulfide exchange, with the

covalent labeling detectable as an increase in the total mass of the protein (as modelled in

Fig. 2C). To selectively target Cys

247

in the screen, we used a mutant t-Aurora A construct

in which the other two cysteines were mutated (C290A and C319V). The choice of amino

acid substitutions for cysteine were chosen based on structural data, with alanine chosen

where valine would be expected to cause steric clashes. A number of cysteine-modifying

compounds were identified (data file S1), from which a subset was selected, based on

diversity of the chemical structures and availability of the compounds, for co-crystallization

trials using the wild-type t-Aurora A construct lacking any cysteine mutations. Crystals were

obtained with compounds 7–80 and 8–34. Unexpectedly, the resulting structures revealed

specific disulfide labeling of Cys

290

within the activation loop two residues C-terminal

to the site of autophosphorylation on Thr

288

, and no modification whatsoever of Cys

247

Both the 7–80– and 8–34–modified structures were highly similar and showed a symmetric

activation segment-swapped dimer, with each of the monomers oriented with their N

terminal lobes pointing in the same direction (Fig. 3, A and B). This is distinct from the

dimer configuration observed for cacodylate-modified t-Aurora A, in which the monomers

are oriented with their N-terminal lobes pointing in orthogonal directions at roughly 90°

relative to each other (Fig. 1B). In particular, the active sites of the 7–80- and 8–34-modified

structures revealed an active DFG-in conformation and have the activation segments of

each kinase well positioned for trans-phosphorylation. For reference, comparison with the

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active Akt kinase domain in complex with a GSK3ß substrate peptide (PDB code 1O6L;

Fig. 3C) (

) shows that the Thr

288

sidechain of each Aurora A monomer in both the

7–80– and 8–34–modified structures is well positioned within the active site of the other

monomer for phosphorylation

in trans

(Fig. 3C). Our structures contrast with a previously

published structure of an unphosphorylated Aurora A kinase domain in complex with a

TPX2 fragment, containing reduced cysteine residues (PDB code 4C3P) (

). Whereas this

unphosphorylated and fully reduced Aurora A-TPX2 complex showed a similar activation

segment-swapped dimerization mode as our 7–80– and 8–34–modified structures, key active

site residues within our 7–80– and 8–34–modified structures are positioned more favorably

for catalysis. The fully reduced Aurora A-TPX2 fragment complex contained a displaced

catalytic base (Glu

l81

) in both monomers, and either a misplaced or disordered region of the

activation loop containing Thr

288

(Fig. 3, D and E). These structural comparisons emphasize

the importance of how the 7–80 and 8–34 disulfide adducts on Cys

290

stabilize a more

trans-phosphorylation–competent conformation within the Aurora A kinase domain dimer

than what was previously observed in the fully reduced Aurora A-TPX2 fragment complex.

These findings point to a previously unrecognized role of C

290

within the activation loop in

promoting autophosphorylation of Aurora A.

Mutation of the activation loop cysteine impairs Aurora A autophosphorylation in

Xenopus

egg extracts and in mammalian cells

To confirm the importance of the activation loop cysteine in promoting Aurora A

autophosphorylation in a more physiologically relevant context, we used the

Xenopus

egg extract system which, as noted above, represents a convenient tool for dissecting the

mechanisms of Aurora A activation. A metaphase-arrested

Xenopus

egg extract was first

depleted of endogenous xAurora A using a bead-immobilized xAurora A-binding fragment

of xCEP192. The extract was then supplemented with recombinant wild-type or mutant,

FLAG-tagged xAurora A proteins (Fig. 4A). xAurora A activation was induced by addition

of demembranated sperm nuclei as a source of centrioles (Fig. 4B) or by addition of a

bivalent anti-xAurora A antibody to artificially induce xAurora A dimerization (Fig. 4C),

and autophosphorylation was monitored as a function of time by SDS-PAGE followed by

immunoblotting. Both centriole-induced clustering and antibody-induced dimerization of

endogenous, wild type FLAG-tagged xAurora A resulted in robust autophosphorylation

on Thr

295

(equivalent to human Thr

288

) (Fig. 4, A and B). No impairment of Thr

295

autophosphorylation was observed for the C254V mutant (equivalent to C247V in human

Aurora A), which actually showed stronger Thr

295

phosphorylation relative to the wild type.

Mutation of the activation loop Cys

297

(equivalent to human Cys

290

), however, completely

abolished autophosphorylation to a similar level as that seen with the non-phosphorylatable

T295A and the D281A kinase-dead mutants (equivalent to human T288A and D274A,

respectively). Similar results were obtained using undepleted

Xenopus

egg extracts, in

which exogenous FLAG-tagged wild-type or mutant xAurora A constructs were selectively

dimerized and activated by addition of an anti-FLAG antibody (Fig. 4D). Notably, the loss

of autophosphorylation observed upon mutation of the activation loop cysteine occurred

despite the fact that

Xenopus

C297A and the human C290A mutants retain catalytic activity

(

–

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Furthermore, analogous results were obtained in mammalian cells. Stable HeLa cell lines

were engineered to express a doxycycline-inducible shRNA to knock down endogenous

Aurora A, and shRNA-resistant FLAG-tagged Aurora A constructs driven by a minimal

fragment of the native Aurora A promoter (

) (Fig. 5A). Both endogenous and wild-type

FLAG-tagged Aurora A showed robust mitotic autophosphorylation on Thr

288

(Fig. 5B).

Consistent with our findings in the

Xenopus

egg extract system, mutation of Cys

290

to alanine completely eliminated Thr

288

phosphorylation, resulting in similar levels of

phosphorylation as those observed upon mutation of the phospho-acceptors within the

activation loop (T288V, and T287V + T288V), or in the D256N + D274N kinase-dead

mutant. In addition, there was also an apparent dominant negative effect of expression of

exogenous Aurora A constructs on endogenous Aurora A Thr

288

phosphorylation. This

likely resulted from exogenous constructs outcompeting endogenous Aurora A for binding

to interactors such as CEP192 and TPX2. These interactors are stoichiometrically less

abundant than Aurora A (

). Together, these results in the

Xenopus

egg extract system

and in mammalian cells indicate an essential physiological role for Cys

290

in Aurora A

autophosphorylation on Thr

288

Oxidative modifications of the activation loop cysteine with coenzyme A (CoAlation) may

promote the formation of a disulfide-linked Aurora A kinase domain homodimer

Oxidative modifications of cysteine side chain thiols are increasingly recognized to

play important physiological roles in regulating protein structure and function (

Consistent with this, our structures of 7–80 and 8–34 modified t-Aurora A indicate

how disulfide modifications of Cys

290

may promote Thr

288

autophosphorylation. To

further evaluate whether oxidative modifications of Cys

290

play a role in Aurora A

autophosphorylation, we tested the effect of adding the reducing agent dithiothreitol (DTT)

to the

Xenopus

egg extract-based assay. Given the already strong reducing environment of

the cytosol, with concentrations of reduced glutathione in the millimolar range (

), we

used a relatively high concentration of DTT in these assays to induce a sufficient redox

perturbation. xAurora A autophosphorylation was markedly inhibited by the addition of

DTT (Fig. 6A), consistent with the involvement of oxidative modification(s) in xAurora A

activation in the context of a cytosolic extract. We cannot, however, completely dismiss the

possibility that other redox-sensitive biochemical processes in the

Xenopus

egg extract may

have been disrupted.

The relevant oxidative modification of Aurora A that occurs

in vivo

is not known.

However, disulfide modification of Aurora A Cys

290

by coenzyme A (CoAlation) was

recently reported as an inhibitory modification that can occur within mammalian cells

under oxidative stress (

). A reported crystal structure of a CoAlated Aurora A kinase

domain shows CoA disulfide bonded to the Cys

290

thiol with the ADP moiety of CoA

inserted into the ATP-binding pocket, accounting for the inhibitory effect of CoAlation on

Aurora A kinase activity (

). We hypothesized that, while CoAlated Cys

290

is inhibitory

in a monomeric Aurora A kinase domain, it might instead promote autophosphorylation

in the context of a kinase domain dimer by stabilizing a more catalytically competent

conformation, similar to what was observed in our 7–80- and 8–34-modified t-Aurora

A structures. To examine this, we CoAlated unphosphorylated wild-type t-Aurora A by

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thiol-disulfide exchange (Fig. 6B), and crystallized this in the presence of AMP-PNP,

with the aim of displacing the ADP moiety of CoA from the ATP-binding pocket. The

structure of this wild-type CoAlated t-Aurora A revealed an activation segment-swapped

dimer, with the ADP moiety of CoA from one molecule tightly bound in the ATP-binding

pocket of the other monomer (Fig. 6C), despite the inclusion of excess AMP-PNP in the

buffers during crystallization and subsequent handling and cryoprotection. This was evident

by strong electron density for the 3-phosphate group in CoA not present in AMP-PNP.

Intriguingly, this dimeric structure shows a more extended arrangement of the swapped

activation segments with the kinase domains compared to what was observed in the

cacodylate-modified dimer structure (Fig. 1B). Our structure markedly contrasts with the

previously reported CoAlated and phosphorylated Aurora A kinase domain structure, which

crystallized as a monomer with CoA bound intramolecularly within the ATP-binding pocket

(

). In addition, in our wild-type unphosphorylated CoAlated t-Aurora A structure, no

electron density was visible for the TPX2 residues fused at the N-terminus, indicating that

this segment is now disordered, which contrasts with all of our other t-Aurora A structures

determined in this study.

We next prepared a CoAlated and non-phosphorylated form of the C247V + C319V

double mutant t-Aurora A for structural analysis. Mass spectrometry data indicated that,

in contrast to the wild-type CoAlated construct, only approximately two thirds of the

double mutant protein was CoAlated (fig. S1). In contrast to the structure obtained with

the wild type unphosphorylated fully CoAlated t-Aurora A, the structure of the C247V +

C319V unphosphorylated CoAlated t-Aurora A construct revealed an activation segment

swapped dimer in which AMP-PNP was bound at the ATP-binding pocket (Fig. 6D). We

did not observe any electron density for CoA. Instead, we observed notable differences

in the conformation of the activation segments compared with the cacodylate-modified

dimer structure (Figs. 6E and 1B), including weak electron density at the dimer interface

consistent with a symmetric disulfide bond between the Cys

290

side chains from the

two molecules within the dimer (Fig. 6F). Of note, the kinase domain in our CoAlated

dimer is also in an inactive DFG-out conformation. Given the incomplete CoAlation of

the protein used for crystallization, it is likely that the small amount of t-Aurora A with

reduced Cys

290

underwent a thiol-disulfide exchange with CoAlated t-Aurora A, resulting

in a subpopulation of the dimers in the crystal consisting of disulfide linked monomers.

This structure obtained using incompletely CoAlated t-Aurora A therefore suggests that

CoAlation may act as a priming modification that facilitates the formation of a symmetric

disulfide dimer of the Aurora A kinase domain.

Dimerization of the Aurora A kinase domain via a symmetric disulfide bond involving the

activation loop cysteine residues promotes autophosphorylation

Because the structure of the C247V + C319V t-Aurora A construct was obtained using

incompletely CoAlated protein, and corresponded to a mixture of predominantly CoAlated

protein with a minor component of the disulfide dimer, we next set out to specifically

crystallize the disulfide linked form. To do this we created a C247V + D256N + C319V

kinase-dead mutant t-Aurora A construct in which Cys

290

was the sole cysteine residue.

We then used Ellman’s reagent to generate a reactive disulfide form of this t-Aurora A

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construct (

), which we reacted with unmodified reduced protein. Thiol-disulfide exchange

between these two forms of the protein resulted in a t-Aurora A homodimer disulfide bonded

via Cys

290

, which was then specifically purified by size-exclusion chromatography. The

crystal structure of this t-Aurora A disulfide homodimer revealed a dimer configuration

very similar to what was observed for our cacodylate-modified and double mutant CoAlated

dimer structures (Fig. 7, A and B). However, whereas the cacodylate-modified and CoAlated

double mutant dimer structures were catalytically inactive with their activation segments

in a DFG-out conformation, this kinase-dead disulfide homodimer structure showed an

active DFG-in conformation (Fig. 7C). The Cys

290

-Cys

290

disulfide within the homodimer

structure showed strong electron density and is positioned at the center of the dimer

interface, similar to what was seen in our CoAlated C247V + C319V dimer structure.

As a consequence, the Thr

288

residue of each monomer is placed at a midpoint position

in between the two active sites and is not optimally positioned for phosphorylation

cis

in trans

. However, the fact that the activation segment is seen in multiple distinct

conformations in the cacodylate-modified, double mutant CoAlated and Cys

290

-Cys

290

disulfide dimer structures suggests that this dimerization mode shared by these forms of

t-Aurora A can accommodate a range of activation segment conformations. We therefore

speculate that additional activation segment conformations would be accessible within this

dimer that would permit movement of each Thr

288

residue into one or both active sites for

phosphorylation.

To directly test this, we examined the ability of the t-Aurora A disulfide dimer to

autophosphorylate

in vitro

. We generated and purified catalytically active disulfide

homodimers of wild-type and a C247V + C319V double mutant t-Aurora A constructs.

Incubation of these disulfide homodimers with ATP resulted in potent autophosphorylation

of the dimer at Thr

288

(upper bands), whereas t-Aurora A monomers (lower bands) did

not show substantial autophosphorylation at the protein concentrations and incubation times

used (Fig. 8A). These findings are consistent with enhanced autophosphorylation occurring

intramolecularly within the disulfide homodimer. It is worth noting that we were unable to

obtain disulfide dimers in t-Aurora A constructs containing the C290A mutation, even when

Cys

247

and Cys

319

were available, in agreement with a report from Tsuchiya et al (

) that

wild type but not C290A Aurora A forms disulfide dimers

in vitro

upon treatment with

. These findings are consistent with Cys

290

being critical for disulfide dimer-enhanced

autophosphorylation.

To determine if the phosphorylation occurred

in trans

in cis

, we heterodimerized

a maltose binding protein (MBP)-tagged kinase-dead construct with a His

-tagged

catalytically active construct. Assay of this disulfide heterodimer after tandem affinity

purification showed potent autophosphorylation as was seen for the disulfide homodimers,

and analysis of the autophosphorylated heterodimer following reduction with DTT

revealed that phosphorylation occurred on both monomers, but predominantly on the

MBP-tagged inactive kinase domain (Fig. 8B), consistent with a strong preference for

trans

-phosphorylation.

Lim et al.

Page 9

Sci Signal

. Author manuscript; available in PMC 2021 October 13.

Author Manuscript