doi.org/10.26434/chemrxiv.7303988.v2 Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and Its Domains and How This Is Modulated upon Interaction with Cdk2/cyclin A Rebecca Beveridge, Lukasz Migas, Richard Kriwacki, Perdita E. Barran Submitted date: 19/12/2018 • Posted date: 19/12/2018 Licence: CC BY-NC 4.0 Citation information: Beveridge, Rebecca; Migas, Lukasz; Kriwacki, Richard; Barran, Perdita E. (2018): Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and Its Domains and How This Is Modulated upon Interaction with Cdk2/cyclin A. ChemRxiv. Preprint. Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states. File list (5) download file view on ChemRxiv beveridge_migas_p27complex_SI_sub.pdf (887.14 KiB) download file view on ChemRxiv beveridge_migas_p27complex_sub.pdf (767.42 KiB) download file view on ChemRxiv beveridge_migas_p27complex_v2_SI.docx (1.01 MiB) download file view on ChemRxiv beveridge_migas_p27complex_graphical_abstract.png (607.02 KiB) download file view on ChemRxiv beveridge_migas_p27complex_v2.docx (1.48 MiB)
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doi.org/10.26434/chemrxiv.7303988.v2
Ion Mobility Mass Spectrometry Measures the ConformationalLandscape of p27 and Its Domains and How This Is Modulated uponInteraction with Cdk2/cyclin ARebecca Beveridge, Lukasz Migas, Richard Kriwacki, Perdita E. Barran
Submitted date: 19/12/2018 • Posted date: 19/12/2018Licence: CC BY-NC 4.0Citation information: Beveridge, Rebecca; Migas, Lukasz; Kriwacki, Richard; Barran, Perdita E. (2018): IonMobility Mass Spectrometry Measures the Conformational Landscape of p27 and Its Domains and How ThisIs Modulated upon Interaction with Cdk2/cyclin A. ChemRxiv. Preprint.
Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding totheir partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficultto visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex isaccompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function,particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformationaldiversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsicdynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwiseapproach reveals the conformational distributions of the constituent proteins and how they are restructured oncomplex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneitycf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini ofp27 interact with Cdk2/cyclin A in multiple closely associated states.
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Figure S1. a) Mass spectra of p27-FL permutants sprayed from 200 mM ammonium acetate solution
(MWcalculated 22340 Da; MWmeasured 22335 Da (p27-FL-14); MWmeasured
22338 Da (p27-FL-31);
MWmeasured 22305 Da (p27-FL-56)).b) Collision cross section distributions as a function of charge state.
The intensity of the individual CCSDs were scaled by taking into account the intensity of the mass
spectrum peak and the area of the CCSDs. Mass spectra are shown in a) were acquired on a Waters
Q-ToF Global whilst the ion mobility profiles represent results from a home-built IM-MS Q-ToF
instrument, hence the charge state distributions differ slightly.
Figure S2. Mass spectra of Cdk2/cyclin A in presence of p27-FL permutants. The mass spectrum shows
wide charge state distribution of the p27-FL as well as peaks of the Cdk2/cyclin A dimer and the
Cdk2/cyclin A/p27-FL trimer.
Figure S3. Collision cross section distributions of individual charge states and the global
conformational landscape of the Cdk2/cyclin A/p27-FL trimeric complex with wild-type (31) and
modified (14 and 56) permutants. The complex was sprayed from 200 mM ammonium acetate at
pH 6.8. The global CCSDs shown in black are the sum of the CCS distributions of individual charges of
the complex.
Materials and Methods
Materials
Truncated human cyclin A (residues 173-432), full-length human Cdk2, p27-C, p27-KID and p27-FL
were provided by Richard Kriwacki, St. Jude Children's Research Hospital, Memphis, TN and were
prepared as reported.[1–3] The purified proteins were buffer exchanged into 200 mM ammonium
acetate at pH 6.8 using Micro Bio-Spin 6 columns (Bio-Rad, Hercules, CA, USA). The Cdk2/cyclin A was
provided as an equimolar dimer and was incubated with p27 constructs for 30 minutes at room
temperature at a 1:1 ratio for experiments on the trimeric complex. All protein samples were prepared
to final concentrations of 15 μM in 200 mM ammonium acetate at pH 6.8.
Nano-electrospray ionisation (nESI)
nESI was employed for all MS and IM-MS experiments. Samples were ionized by applying a positive
potential of 1.6 kV through a thin platinum wire (diameter 0.125 mm, Goodfellow, Huntingdon, UK)
inserted into a nESI tips that were pulled in-house from thin-walled glass capillaries (i.d. 0.9 mm, o.d.
1.2 mm, World Precision Instruments, Stevenage, UK) using Flaming/Brown micropipette puller
(Sutter Instrument Co., Novato, CA).
Mass Spectrometry
All MS experiments were performed on a Q-ToF Global (Waters, Manchester, UK) with sampling cone
voltage of 60 V, source pressure 3.3 mbar, source temperature of 100 ˚C, collision cell pressure of 6 ×
10-3 mbar and collision energy of 5 V.
Ion mobility-mass spectrometry
IM-MS experiments were carried out on a Waters Q-ToF I instrument that was modified in-house to
include a 5.1 cm drift tube which has been described elsewhere.[4] The temperature and pressure of
helium in the drift cell were approximately 28˚C and 4 Torr respectively. Measurements were made
at 6 different drift voltages from 60 V to 20 V. The precise pressure and temperature was recorded for
every drift voltage and used in the calculations of CCSs. Each experiment was performed in triplicate.
Ion arrival time distributions were recorded by synchronisation of the release of ions into the drift cell
with mass spectral acquisition. The CCS distribution plots are derived from raw arrival time data using
Equation 1 below.[5]
Ωavg = (18π)1/2
16[
1
𝑚𝑏+
1
𝑚]
1/2 𝑧𝑒
(𝐾𝐵𝑇)1/2
1
𝜌
𝑡𝑑𝑉
𝐿2 Equation 1
Where m and mb are the masses of the ion and buffer gas, respectively; z is the ion charge state; e is
the elementary charge; KB is the Boltzmann constant; T is the gas temperature; ρ is the buffer gas
density; L is the drift tube length; V is the voltage across the drift tube; and td is the drift time.
The raw arrival time output (ta) includes time the ions spend outside of the drift cell but within the
mass spectrometer, known as the dead time (t0). The value for t0 is calculated by taking an average
value of the intercept from a linear plot of average arrival time versus pressure/temperature and is
subtracted from the arrival time to calculate drift time (tD):
tD = ta – t0 Equation 2
All MS and IM-MS data were analysed using Masslynx v4.1 software (Waters, Manchester, UK),
ORIGAMIANALYSE, Origin v8.5 (Originlab Corporation, USA) and Microsoft Excel.[6] Mass spectra were
deconvoluted in ORIGAMIANALYSE using the UniDec deconvolution algorithm.[6,7]
Modelling of CCS framework boundaries
The theoretical lower and upper boundaries of the collision cross section distribution were calculated
using the Beveridge-Barran framework model. In the model, the lower boundary is predicted by
assuming the protein adopts a highly compact, sphere-like conformation for which CCS can be
calculated using the following equation:
𝐶𝐶𝑆𝑙𝑜𝑤𝑒𝑟 = 𝜋 (3𝑉
4𝜋)
23⁄
× 1.19 Equation 3
where V is the volume of protein sphere which can be calculated using equation 4:
𝑉 = 𝑀𝑊
𝜌 Equation 4
in which MW is the molecular weight of the protein and ρ is the average protein density (0.904 Da/Å3).
Conversely, the upper CCS boundary is predicted for a protein structure that adopts a fully extended,
cylinder-like conformation. In this case, the rotationally averaged CCS can be calculated using equation
5:
𝐶𝐶𝑆𝑢𝑝𝑝𝑒𝑟 = (4
𝜋) 𝑟𝑙 + 2𝑟2 × 1.19
where l is the length of the cylinder and r is its radius. For full detail about modelling the lower and
upper CCS boundaries, please refer to the original publication.[8]
Calculation of CCSs from crystal structure
X-ray crystallography structure of Cdk2/cyclin A/p27-C (1JSU) was downloaded from the RSCB Protein
Data Bank (PDB) and hydrogen atoms were added to the structure using XLEaP module in the AMBER
Software Suite.[9] The structure was subsequently minimised in vacuo using AMBER with the amber
ff99SB force field. Collision cross section was calculated using the trajectory method available in
MOBCAL.[10]
[1] E. R. Lacy, I. Filippov, W. S. Lewis, S. Otieno, L. Xiao, S. Weiss, L. Hengst, R. W. Kriwacki, Nat. Struct. Mol. Biol. 2004, 11, 358–364.
[2] M. Grimmler, Y. Wang, T. Mund, Z. Cilenšek, E. M. Keidel, M. B. Waddell, H. Jäkel, M. Kullmann, R. W. Kriwacki, L. Hengst, Cell 2007, 128, 269–280.
[3] R. K. Das, Y. Huang, A. H. Phillips, R. W. Kriwacki, R. V Pappu, Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 5616–5621.
[4] B. J. McCullough, J. M. D. Kalapothakis, H. Eastwood, P. Kemper, D. MacMillan, K. Taylor, J. Dorin, P. E. Barran, Anal. Chem. 2008, 80, 6336–6344.
[5] M. F. Jarrold, Accounts Chem. Res. 1999, 32, 360–367.
[6] L. G. Migas, A. P. France, B. Bellina, P. E. Barran, Int. J. Mass Spectrom. 2017, 427, 20–28.
[7] M. T. Marty, A. J. Baldwin, E. G. Marklund, G. K. A. Hochberg, J. L. P. Benesch, C. V Robinson, Anal. Chem. 2015, 87, 4370–4376.
[8] R. Beveridge, S. Covill, K. J. Pacholarz, J. M. D. Kalapothakis, C. E. MacPhee, P. E. Barran, Anal. Chem. 2014, 86, 10979–10991.
[9] D. A. Case, T. E. Cheatham, T. Darden, H. Gohlke, R. Luo, K. M. Merz, A. Onufriev, C. Simmerling, B. Wang, R. J. Woods, J. Comput. Chem. 2005, 26, 1668–1688.
[10] M. F. Mesleh, J. M. Hunter, A. A. Shvartsburg, G. C. Schatz, M. F. Jarrold, J. Phys. Chem. 1996, 3654, 16082–16086.
download fileview on ChemRxivbeveridge_migas_p27complex_SI_sub.pdf (887.14 KiB)
interactions are suitable for signalling cascades as they ensure reversibility such that a signal can
be turned off promptly.
The p27 protein is composed of two distinct domains, and in the presence of Cdk2/cyclin A, the
N-terminal kinase-inhibitory domain (KID) sequentially folds into a conformation that blocks
substrate binding to cyclin A and independently inhibits ATP binding to Cdk2 and substrate
phosphorylation.[3] The C-terminal domain of p27, which contains several sites for post-
translational modification, has been shown by NMR to be highly disordered in isolation and to
exhibit disorder within its C-terminal domain (p27-C) when the KID is bound to Cdk2/cyclin A.[3]
The flexibility of p27-C within the complex allows it to be phosphorylated, resulting in its ubiquitin-
dependent proteolysis and subsequent reactivation of the Cdk2/cyclin A complex and cell cycle
progression.[3] Molecular dynamics simulations further demonstrated that the segment of p27-C
immediately following the KID protrudes at an abrupt angle perpendicular to the surface of
Cdk2/cyclin A.[3] This is thought to arise due to electrostatic repulsion between residues 110-140
of p27 and the negatively charged surface of Cdk2/cyclin A/p27-KID, hence causing the more
distal segment of p27-C to assume highly disordered conformations. This is consistent with
analytical ultracentrifugation and small angle X-ray scattering experiments that confirm a highly
extended conformation of p27-C when part of the ternary complex.[4]
The objectives of this study were to examine the dynamic behaviour of each domain of p27, of
the intact WT protein and as part of a trimeric complex with Cdk2/cyclin A. In parallel, an
investigation of the C terminal permutants of p27 has been conducted.[5] In this work, we sought
to examine the conformational behavior of the N-terminal KID which folds upon binding and the
C-terminal domain which remains disordered. The conformational distributions of full-length p27
(p27-FL) were measured, as well as p27-KID and p27-C which were expressed as separate
proteins. We show that the binding of p27-KID to Cdk2/cyclin A significantly increases the
conformational flexibility of the protein complex and that the binding of p27-FL has an even greater
effect of the dynamic behaviour of the complex. A cartoon representation of the complexes that
were analyzed are depicted in Figure 1.
3
Figure 1 Schematic representation of the Cdk2/cyclin A dimer, Cdk2/cyclin A/p27-KID trimer and
Cdk2/cyclin A/p27-FL trimer.
The conformational distribution analysis of the isolated p27 constructs and their Cdk2/cyclin A
trimeric complex was performed using an ion mobility mass spectrometer which allows direct
measurement of rotationally averaged collision cross sections (DTCCSHe).[6] IM-MS is a hybrid
method permitting separation of gas phase ions according to their mobility and subsequently their
mass-to-charge ratio. Ions were generated using nanoelectrospray ionization (nESI) which allows
retention of the native fold of the protein as they are transferred into the gas phase; following
desolvation, ions are separated in the ion mobility cell according to their velocity under a weak
electric field while they experience collisions with an inert buffer gas. The mobility of an ion is
dependent upon its size, shape and charge. In contrast to X-ray crystallography and NMR
spectroscopy, IM-MS cannot provide atomistic detail about the samples; however, it can be
employed to study conformationally dynamic systems and heterogeneic stoichiometries in a
single experiment.
4
Figure 2 The p27-FL intrinsically disordered protein is composed of two domains, N-terminal p27-
KID and the C-terminal p27-C. The individual domains, as well as the full-length p27 protein, were
investigated using MS and IM-MS methods to elucidate their structural features while isolated in
the gas phase. Protein samples were sprayed from 200 mM ammonium acetate, pH 6.8. (a) A
representative sequence of the p27-FL components, namely p27-KID, p27-C and p27-FL. Native
mass spectra and collision cross section distributions (CCSDs) of individual proteins are shown
in (b) p27-C (MWcalculated 11234 Da, MWmeasured 11234 Da); (c) p27-KID (MWcalculated 10447 Da,
MWmeasured 10444 Da); and (d) p27-FL (MWcalculated 22340 Da, MWmeasured 22338 Da), respectively.
The mass spectra show the charge state distributions of each protein whilst the ion mobility
profiles highlight the structural heterogeneity of individual charge states. The height of the CCSD
curve is proportional to the height of the corresponding peak in the mass spectrum and the area
of the ion mobility peak.
In order to assess the level of disorder exhibited by the p27 proteins, we examined the charge
state distribution (CSD) from their native mass spectra and compared them against the theoretical
maximum charge state of a globular protein of the same molecular weight (MW). The maximum
number of positive charges a globular protein can accommodate can be calculated using the
following equation[7]
𝑍𝑅 = 0.0778√𝑚
5
where ZR is the maximum charge and m is the molecular weight of the protein. Focusing on the
p27-C domain first, the MS analysis reveals a CSD ranging from [M+6H]6+ to [M+17H]17+, with
most intensity based in the lowest two charge states, [M+6H]6+ and [M+7H]7+ (Figure 2b), which
is indicative of a protein that contains both compact (perhaps structured) as well as extended
regions.[8] The ZR of p27-C is 8.24, meaning that the maximum number of charges a globular form
of the protein could carry is 8 and all observed charge states above this threshold (from [M+9H]9+
to [M+17H]17+) must be therefore extended to some degree. In addition to the broad CSD, the
collision cross section distributions (CCSDs) from the ion mobility experiments confirm that charge
states below the ZR limit have a compact geometry, with CCS ranging from 8-15 nm2. Ions with
charge states above [M+7H]7+ increase in size, up to 28 nm2 for [M+17H]17+. The overall baseline
to baseline CCSD (CCSDBB) is 8-28 nm2 (ΔCCSDBB 20 nm2). In comparison to p27-C, the CSD
and CCSD of p27-KID (Figure 2c) suggest a conformational ensemble as flexible as that of p27-
C, which is surprising considering the KID domain, which folds upon binding to Cdk2/cyclin A, has
been shown to adopt partially populated secondary structure within different sub-domains in
isolation prior to binding[9,10] The low charge states of [M+6H]6+ and [M+7H]7+ of p27-KID have
CCSDs ranging from 7.5-15 nm2 and 8-18 nm2 respectively, both of which are resolved into two
distinct populations, with the larger conformational family of [M+6H]6+ overlapping in size with the
smaller family of [M+7H]7+. The ZR of p27-KID is 7.95, suggesting that all charge states above
[M+7H]7+ arise from partially extended and disordered conformations. A relatively high proportion
of ions are present above the ZR limit, which results in a broad CCSDBB, ranging from 7.5-25 nm2
and ΔCCSDBB 17.5 nm2.
The relationship between charge and CCS for p27-KID is unusual but reproducible. The smaller
conformational family of [M+6H]6+ is centered around 9.5 nm2, which is smaller than the average
CCS of [M+5H]5+ (11.3 nm2). This decrease in CCS upon the addition of a proton could indicate
to stabilization of compact conformations. Also surprising is the gradual decrease in average CCS
for increasing charge states above [M+10H]10+. A region of the KID domain has the propensity to
form a helix, and a possible explanation for the lack of extension in the higher charge states is
that the conformations giving rise to these highly charged ions are helical. In such a conformation
protonated residues would be held away from each other, minimizing coulombic repulsion, which
would result in no or little increase in CCS with respect to increased protonation.[11,12] This would
indicate that the lower charge states are more charge solvated forms, which is supported by the
substantial adductation of the protein by sodium and potassium for z=5-7, and the fact that a
6
substantial portion of the measured conformational spread comes from charges states that fall
below the limit predicted for compact/folded states.[7]
The native mass spectrum of p27-FL (Figure 2d) exhibits a wide CSD from [M+9H]9+ to
[M+33H]33+; the multimodal nature of the CSD reveals the coexistence of multiple distinct
conformational families upon desolvation.[13] The majority of these highly charged species, fall
well above the ZR limit of 11.6 and in combination with the broad CCSDBB of 13-55 nm2
(ΔCCSDBB 42 nm2) it is evident that p27-FL forms a myriad of extended states. The ATDs of
individual charge states, in particular [M+20H]20+ to [M+23H]23+ indicate multiple conformational
families are sampled from solution which span nearly the entire width of the CCSD. In previous
work, we have demonstrated how the positioning of charged amino acid residues of the p27-C
domain substantially affects the populations of stable conformers. A charge permutant of p27-C
(termed p27-C-14) in which charged residues are more evenly dispersed than within the wild-
type sequence, was previously found to populate available conformational space more evenly
than the wild-type (p27-C-31), which in turn occupies significantly more space than a second
permutant (p27-C-56), in which oppositely charged residues are partially sequestered within the
sequence.[4] This effect is mitigated for the full-length protein while the two p27-C permutants, and
the corresponding wild-type construct (p27-C-31), display a broadly similar conformational
landscape (Figure 2 and S1).[4]
The interaction between the p27 domains and Cdk2/cyclin A was then investigated. Native MS
results confirm the stoichiometry of the protein complexes; Cdk2/cyclin A alone exists only as a
dimer which is predominantly present in three charge states [M+15H]15+-[M+17H]17+, all below the
ZR limit of 19.75. Upon addition of p27-KID, a trimeric complex is observed, in four charge states,
[M+16H]16+-[M+19H]19+ (ZR=21.29). On addition of p27-FL, the Cdk2/cyclin A/p27-FL is observed,
which displays four charge states, [M+18H]18+-[M+21H]21+ (ZR=22.92), with an overall increase in
net charge of only 2 cf. the KID complex.
In the mass spectrum of the p27-FL (WT) complex, a small quantity of the unbound Cdk2/cyclin
A dimer is observed, as well as some cyclin A/p27-FL complex, attributed to displacement of a
small amount of Cdk2 upon p27 binding to Cdk2/cyclin A. This provides details on the mechanism
of complex formation which complements previous results: Lacy et al. reported that binding of
p27-KID to cyclin A alone is more thermodynamically favourable than binding Cdk2 alone.[9]
Furthermore, binding of p27 to the Cdk2/cyclin A complex occurs at a similar rate to that observed
for binding to cyclin A alone. This is justified by slow remodelling of Cdk2 that occurs upon
7
association with p27. While considering the formation of the Cdk2/cyclin A/p27-FL complex, we
also examined the behaviour of trimeric complexes containing full length p27 in which the charged
residues in the C-terminus were redistributed as in the p27-C-14 and p27-C-56 permutants
(p27-FL-14 and p27-FL-56, respectively); in both cases, the complex is formed and present in
[M+18H]18+-[M+21H]21+ charge states, albeit the relative intensity of these has changed (shown in
Figure S2). The proportion of the trimeric complex is greatest for wild-type p27-FL (termed p27-
FL-31) than for the 14 and 56 permutants; p27-FL-31 is the endogenous binding partner of
Cdk2/cyclin A, and this suggests that the C-terminal domain forms interactions that stabilize the
trimeric complex that are less favourable or absent in complexes with the two charge permutants.
Figure 3 MS and IM-MS investigation of Cdk2/cyclin A complex in the absence and presence of
p27-KID and p27-FL. Proteins were sprayed from 200 mM ammonium acetate at pH 6.8. (a) The
Cdk2/cyclin A complex (MWcalculated 64465 Da, MWmeasured 64644 Da) appears predominantly in
three charge states, all of which have a narrow CCSDs; (b) in the presence of p27-KID, a ternary
complex forms (MWcalculated 74912 Da, MWmeasured 74981 Da), shown in four charge states of which
CCSDs suggest a majority of the complex is formed with compact states of p27-KID. The apex of
the distribution is only marginally larger than that of dimeric Cdk2/cyclin A (40.1 nm2 and 38.5
nm2), whilst the width of the distribution highlights a significant increase in conformational
heterogeneity. (c) As with p27-KID, the addition of p27-FL results in the formation of Cdk2/cyclin
A/p27-FL (MWcalculated 86796 Da, MWmeasured 86822 Da) complex, predominantly present in four
charge states; the cross-sectional distribution widens further, yet the apex of the distribution only
8
increased to 43.6 nm2. The global CCSDs shown in black are the sums of the CCS distributions
of individual charges of the complex.
Table 1 Summary of IM-MS findings for Cdk2/cyclin A, Cdk2/cyclin A/p27-KID and Cdk2/cyclin
A/p27-FL complex.
Species CCSDBB
(nm2) ΔCCSDBB
(nm2) Apex of global CCSD
(nm2)
Cdk2/cyclin A 32 – 47 15 38.5
Cdk2/cyclin A/p27-KID 32 – 62 30 40.1
Cdk2/cyclin A/p27-FL 35 – 70 35 43.6
In all cases, the CCSDBB was calculated by measuring the width of the CCSD at the baseline.
The CCSDs of individual charge states of the complexes are shown in Figure 3, whilst the
combined global overview of the behaviour of the protein is shown in Figure 4 and summarized
in Table 1. Viewing the ion mobility data as total CCSDs allows a comparison of structural features
of the Cdk2/cyclin A complex, the isolated p27 domains and how each are modulated in the
trimeric complex. Firstly, the Cdk2/cyclin A (64.5 kDa) dimer adopts a compact conformation
whilst in the gas phase, as illustrated by the narrow ΔCCSDBB of 15 nm2 (32 – 47 nm2). In fact,
the width of the distribution is even narrower than for other natively structured proteins of similar
molecular weight e.g. transthyretin tetramer (55 kDa) or avidin tetramer (66 kDa) which have
ΔCCSDBB of 22 and 20 nm2, respectively, suggestive of a highly ordered complex.[14] The apex
of the CCSD marginally increases with each charge state, centering at 38.5 nm2 in the global
CCSD (Figure 4d), indicating a very rigid quaternary structure with minimal conformational
dynamics, and an upper charge state sitting well below the limit introduced by de La Mora that
may maintain a compact globular geometry.[7]
9
Figure 4 Global CCSDs of isolated p27 domains, dimeric Cdk2/cyclin A complex in the absence
and presence of p27-KID/p27-FL. (a) p27-C, (b) p27-KID, and (c) p27-FL, as well as the protein
complexes, (d) Cdk2/cyclin A, (e) Cdk2/cyclin A/p27-KID and (f) Cdk2/cyclin A/p27-FL. The
vertical dashed lines in plots a-c represent the theoretical CCSDs of the p27 constructs, as
calculated using a framework model from Beveridge et al.[8] The arrows shown in plots a-c mark
the maximum measured CCS for the charge states below the ZR limit, indicative of
globular/structured conformations as described in the text; charge states measured in d-f fall
below the ZR limit hence are not shown. The global CCSDs are the sum of the CCS distributions
of individual charge states of each protein.
Upon addition of p27-KID to Cdk2/cyclin A, the lower boundary of the CCSD remains the same
at 32 nm2, while the upper boundary increases considerably to 62 nm2 (ΔCCSDBB 30 nm2, Figure
4e). The negligible effect of p27-KID on the size of the structure at the lower end of the CCSD
10
indicates that the IDP is capable of tightly binding to the globular dimer. The apex of the global
CCSD is at 40.1 nm2, only 1.6 nm2 larger than that of the Cdk2/cyclin A alone, indicating that
much of the Cdk2/cyclin A/p27-KID complex is found in a compact conformation, although the
apex of these CCSD increases for the complex by 32% (cf. with a 16% increase expected due to
the increase in molecular weight). Such an increase in the upper limit of the CCSD is unlikely to
be achieved by the bound p27-KID alone, rather, we propose that it is due to structural remodelling
exerted by p27 on Cdk2, as discussed by Lacy et al.[9] and Russo et al.[15]
When p27-FL forms the ternary complex with Cdk2/cyclin A, the lower boundary of the CCSD is
still only marginally larger than that of the Cdk2/cyclin A dimer (+3 nm2), whereas the upper
boundary increases to 70 nm2 (ΔCCSDBB 35 nm2, Figure 4f). Incorporation of p27-FL into the
complex increases its size only slightly more than the addition of p27-KID. The apex of the global
CCSD increases by 5.1 nm2 compared to the Cdk2/cyclin A dimer and 3.5 nm2 compared to the
Cdk2/cyclin A/p27-KID complex. In addition, the width of the CCSD of the Cdk2/cyclin A/p27-FL
complex is only marginally larger than that of the p27-FL protein alone (CCSDBB 15-55 nm2,
Figure 4c), despite the complex being three-times the molecular weight of the isolated protein.
We do not attribute this increase in the width of the CCSD to an untethered C terminal domain
(Figure 1); such a configuration would result in a wider CSD, with higher charge states due to the
increased solvent accessibility of the p27-C similar to that found for lymphotactin.[16]
The 14 and 56 permutants also form complexes with wide CCSDs and narrow CSDs, where
the apex of the global distribution shifts from 38.5 nm2 to 50.5 nm2 (+12 nm2) and 47.4 nm2 (+8.9
nm2), respectively. Despite a slight shift in the apex of the global conformation of the complex, the
width and form of the CCSD is nearly identical (as shown in Figure S3). This suggests the
formation of a fuzzy complex upon binding of p27-KID where the C-terminal domain of p27-FL
lies on the surface of the globular protein, retaining some conformational flexibility w.r.t. to
Cdk2/cyclin A for each permutant, irrespective of the charge patterning of the C terminus – which
in isolation is conformationally distinct.[5] The observed narrow range of charges coupled with a
wide spread of conformations is similar to that found for monoclonal antibodies[14] and is attributed
to flexible interactions between domains.[17]
In summary, we have demonstrated that the dynamic propensity of p27-FL is significantly reduced
upon binding to the binary complex of Cdk2/cyclin A, in agreement with the ‘folding-upon-binding’
process that has been shown previously.[9] Additionally, our results show a compact conformation
of the Cdk2/cyclin A dimeric complex, which gains significant conformational flexibility upon
formation of the ternary complex containing p27-KID domain, and only slightly more upon binding
11
of the p27-FL protein. Only subtle changes in the conformational dynamics of the complex were
observed when bound to the 14 and 56 permutants. Overall, we were able to monitor the
dynamic behaviour of individual proteins and when bound in a multimeric complex, while also
extracting structural information.
Acknowledgements
This research was supported by the BBSRC (awards: BB/L015048/1, BB/K017802/1 and
BB/H013636/1 and the BBSRC/EPSRC-funded Manchester Synthetic Biology Research Centre,
SYNBIOCHEM (BB/M017702/1)). BBSRC, EPSRC, Waters Corp., LGC Ltd. and the Universities
of Edinburgh and Manchester are thanked for their support of studentships to RB and LGM. RWK
acknowledges support from the National Cancer Institute (P30CA21765 to St. Jude Children’s
Research Hospital) and ALSAC.
Keywords: mass spectrometry, protein structure, intrinsically disordered proteins
References
[1] L. Hengst, V. Dulic, J. M. Slingerland, E. Lees, S. I. Reed, Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 5291–5295.
[2] K. Polyak, M. H. Lee, H. Erdjument-Bromage, A. Koff, J. M. Roberts, P. Tempst, J. Massagué, Cell 1994, 78, 59–66.
[3] C. A. Galea, A. Nourse, Y. Wang, S. G. Sivakolundu, W. T. Heller, R. W. Kriwacki, T. Heller, R. W. Kriwacki, J. Mol. Biol. 2008, 376, 827–838.
[4] R. K. Das, Y. Huang, A. H. Phillips, R. W. Kriwacki, R. V Pappu, Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 5616–5621.
[5] R. Beveridge, L. G. Migas, R. K. Das, R. V. Pappu, R. W. Kriwacki, P. E. Barran, manuscript submitted
[6] B. J. McCullough, J. M. D. Kalapothakis, H. Eastwood, P. R. Kemper, D. MacMillan, K. Taylor, J. Dorin, P. E. Barran, Anal. Chem. 2008, 80, 6336–6344.
[7] J. Fernandez de la Mora, Anal. Chim. Acta 2000, 406, 93–104.
[8] R. Beveridge, S. Covill, K. J. Pacholarz, J. M. D. Kalapothakis, C. E. Macphee, P. E. Barran, Anal. Chem. 2014, 86, 10979–10991.
[9] E. R. Lacy, I. Filippov, W. S. Lewis, S. Otieno, L. Xiao, S. Weiss, L. Hengst, R. W. Kriwacki, Nat. Struct. Mol. Biol. 2004, 11, 358–364.
[10] S. G. Sivakolundu, D. Bashford, R. W. Kriwacki, J. Mol. Biol. 2005, 353, 1118–1128.
[11] Y. V Berezovskaya, M. Porrini, P. E. Barran, Int. J. Mass Spectrom. 2013, 345, 8–18.
[12] J. M. D. Kalapothakis, Y. V Berezovskaya, C. G. Zampronio, P. A. Faull, P. E. Barran, H. J. Cooper, Chem. Commun. 2014, 50, 198–200.
[13] A. J. Borysik, D. Kovacs, M. Guharoy, P. Tompa, J. Am. Chem. Soc. 2015, 137, 13807–13817.
[14] K. J. Pacholarz, M. Porrini, R. A. Garlish, R. J. Burnley, R. J. Taylor, A. J. Henry, P. E. Barran, Angew. Chemie - Int. Ed. 2014, 53, 7765–7769.
[15] A. A. Russo, P. D. Jeffrey, N. P. Pavletich, Nat. Struct. Biol. 1996, 3, 696–700.
[16] S. R. Harvey, M. Porrini, A. Konijnenberg, D. J. Clarke, R. C. Tyler, P. R. R. Langridge-Smith, C. E. MacPhee, B. F. Volkman, P. E. Barran, J. Phys. Chem. B 2014, 118, 12348–12359.
[17] D. Stuchfield, P. E. Barran, Curr. Opin. Chem. Biol. 2018, 42, 177–185.
download fileview on ChemRxivbeveridge_migas_p27complex_sub.pdf (767.42 KiB)
Figure S1. a) Mass spectra of p27-FL permutants sprayed from 200 mM ammonium acetate solution
(MWcalc 22340 Da; MWexp 22335 Da (p27-FL-14); MWexp
22338 Da (p27-FL-31); MWexp 22305 Da
(p27-FL-56)).b) Collision cross section distributions as a function of charge state. The intensity of the
individual CCSDs were scaled by taking into account the intensity of the mass spectrum peak and the
area of the CCSDs. Mass spectra are shown in a) were acquired on a Waters Q-ToF Global whilst the
ion mobility profiles represent results from a home-built IM-MS Q-ToF instrument, hence the charge
state distributions differ slightly.
Figure S2. Mass spectra of Cdk2/cyclin A in presence of p27-FL permutants. The mass spectrum
shows wide charge state distribution of the p27-FL as well as peaks of the Cdk2/cyclin A dimer and
the Cdk2/cyclin A/p27-FL trimer.
Figure S3. Collision cross section distributions of individual charge states and the global
conformational landscape of the Cdk2/cyclin A/p27-FL trimeric complex with wild-type (31) and
modified (14 and 56) permutants. The complex was sprayed from 200 mM ammonium acetate at
pH 6.8. The global CCSDs shown in black are the sum of the CCS distributions of individual charges of
the complex.
Materials and Methods
Materials
Truncated human cyclin A (residues 173-432), full-length human Cdk2, p27-C, p27-KID and p27-FL
were provided by Richard Kriwacki, St. Jude Children's Research Hospital, Memphis, TN and were
prepared as reported.[1–3] The purified proteins were buffer exchanged into 200 mM ammonium
acetate at pH 6.8 using Micro Bio-Spin 6 columns (Bio-Rad, Hercules, CA, USA). The Cdk2/cyclin A was
provided as an equimolar dimer and was incubated with p27 constructs for 30 minutes at room
temperature at a 1:1 ratio for experiments on the trimeric complex. All protein samples were
prepared to final concentrations of 15 μM in 200 mM ammonium acetate at pH 6.8.
Nano-electrospray ionisation (nESI)
nESI was employed for all MS and IM-MS experiments. Samples were ionized by applying a positive
potential of 1.6 kV through a thin platinum wire (diameter 0.125 mm, Goodfellow, Huntingdon, UK)
inserted into a nESI tips that were pulled in-house from thin-walled glass capillaries (i.d. 0.9 mm, o.d.
1.2 mm, World Precision Instruments, Stevenage, UK) using Flaming/Brown micropipette puller
(Sutter Instrument Co., Novato, CA).
Mass Spectrometry
All MS experiments were performed on a Q-ToF Global (Waters, Manchester, UK) with sampling cone
voltage of 60 V, source pressure 3.3 mbar, source temperature of 100 C̊, collision cell pressure of 6 ×
10-3 mbar and collision energy of 5 V.
Ion mobility-mass spectrometry
IM-MS experiments were carried out on a Waters Q-ToF I instrument that was modified in-house to
include a 5.1 cm drift tube which has been described elsewhere. [4] The temperature and pressure of
helium in the drift cell were approximately 28C̊ and 4 Torr respectively. Measurements were made at
6 different drift voltages from 60 V to 20 V. The precise pressure and temperature was recorded for
every drift voltage and used in the calculations of CCSs. Each experiment was performed in triplicate.
Ion arrival time distributions were recorded by synchronisation of the release of ions into the drift
cell with mass spectral acquisition. The CCS distribution plots are derived from raw arrival time data
using Equation 1 below.[5]
Ωavg=(18 π )1/2
16 [ 1mb
+ 1m ]
1 /2ze
(K BT )1/21ρ
t dV
L2
Equation 1
Where m and mb are the masses of the ion and buffer gas, respectively; z is the ion charge state; e is
the elementary charge; KB is the Boltzmann constant; T is the gas temperature; ρ is the buffer gas
density; L is the drift tube length; V is the voltage across the drift tube; and td is the drift time.
The raw arrival time output (ta) includes time the ions spend outside of the drift cell but within the
mass spectrometer, known as the dead time (t0). The value for t0 is calculated by taking an average
value of the intercept from a linear plot of average arrival time versus pressure/temperature and is
subtracted from the arrival time to calculate drift time (tD):
tD = ta – t0 Equation 2
All MS and IM-MS data were analysed using Masslynx v4.1 software (Waters, Manchester, UK),
ORIGAMIANALYSE, Origin v8.5 (Originlab Corporation, USA) and Microsoft Excel.[6] Mass spectra were
deconvoluted in ORIGAMIANALYSE using the UniDec deconvolution algorithm.[6,7]
Modelling of CCS framework boundaries
The theoretical lower and upper boundaries of the collision cross section distribution were calculated
using the Beveridge-Barran framework model. In the model, the lower boundary is predicted by
assuming the protein adopts a highly compact, sphere-like conformation for which CCS can be
calculated using the following equation:
CCS lower=π ( 3V4 π )
23×1.19 Equation 3
where V is the volume of protein sphere which can be calculated using equation 4:
V=MW
ρ Equation 4
in which MW is the molecular weight of the protein and ρ is the average protein density (0.904
Da/Å3). Conversely, the upper CCS boundary is predicted for a protein structure that adopts a fully
extended, cylinder-like conformation. In this case, the rotationally averaged CCS can be calculated
using equation 5:
CCSupper=( 4π )rl+2 r2×1.19
where l is the length of the cylinder and r is its radius. For full detail about modelling the lower and
upper CCS boundaries, please refer to the original publication.[8]
Calculation of CCSs from crystal structure
X-ray crystallography structure of Cdk2/cyclin A/p27-C (1JSU) was downloaded from the RSCB Protein
Data Bank (PDB) and hydrogen atoms were added to the structure using XLEaP module in the AMBER
Software Suite.[9] The structure was subsequently minimised in vacuo using AMBER with the amber
ff99SB force field. Collision cross section was calculated using the trajectory method available in
MOBCAL.[10]
[1] E. R. Lacy, I. Filippov, W. S. Lewis, S. Otieno, L. Xiao, S. Weiss, L. Hengst, R. W. Kriwacki, Nat. Struct. Mol. Biol. 2004, 11, 358–364.
[2] M. Grimmler, Y. Wang, T. Mund, Z. Cilenšek, E. M. Keidel, M. B. Waddell, H. Jäkel, M. Kullmann, R. W. Kriwacki, L. Hengst, Cell 2007, 128, 269–280.
[3] R. K. Das, Y. Huang, A. H. Phillips, R. W. Kriwacki, R. V Pappu, Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 5616–5621.
[4] B. J. McCullough, J. M. D. Kalapothakis, H. Eastwood, P. Kemper, D. MacMillan, K. Taylor, J. Dorin, P. E. Barran, Anal. Chem. 2008, 80, 6336–6344.
[5] M. F. Jarrold, Accounts Chem. Res. 1999, 32, 360–367.
[6] L. G. Migas, A. P. France, B. Bellina, P. E. Barran, Int. J. Mass Spectrom. 2017, 427, 20–28.
[7] M. T. Marty, A. J. Baldwin, E. G. Marklund, G. K. A. Hochberg, J. L. P. Benesch, C. V Robinson, Anal. Chem. 2015, 87, 4370–4376.
[8] R. Beveridge, S. Covill, K. J. Pacholarz, J. M. D. Kalapothakis, C. E. MacPhee, P. E. Barran, Anal. Chem. 2014, 86, 10979–10991.
[9] D. A. Case, T. E. Cheatham, T. Darden, H. Gohlke, R. Luo, K. M. Merz, A. Onufriev, C. Simmerling, B. Wang, R. J. Woods, J. Comput. Chem. 2005, 26, 1668–1688.
[10] M. F. Mesleh, J. M. Hunter, A. A. Shvartsburg, G. C. Schatz, M. F. Jarrold, J. Phys. Chem. 1996, 3654, 16082–16086.
download fileview on ChemRxivbeveridge_migas_p27complex_v2_SI.docx (1.01 MiB)
[8] L. Testa, S. Brocca, C. Santambrogio, A. D’Urzo, J. Habchi, S. Longhi, V. N. Uversky, R.Grandori, Intrinsically Disord. proteins 2013, 1, e25068.
[9] K. C. B. De Freitas, J. Am. Soc. Mass Spectrom. 2018, 29, 2059–2066.
[10] E. R. Lacy, I. Filippov, W. S. Lewis, S. Otieno, L. Xiao, S. Weiss, L. Hengst, R. W.Kriwacki, Nat. Struct. Mol. Biol. 2004, 11, 358–364.
[11] S. G. Sivakolundu, D. Bashford, R. W. Kriwacki, J. Mol. Biol. 2005, 353, 1118–1128.
[12] Y. Berezovskaya, M. Porrini, P. E. Barran, Int. J. Mass Spectrom. 2013, 345–347, 8–18.
[13] J. M. D. Kalapothakis, Y. V Berezovskaya, C. G. Zampronio, P. A. Faull, P. E. Barran, H.J. Cooper, Chem. Commun. 2014, 50, 198–200.
[14] A. J. Borysik, D. Kovacs, M. Guharoy, P. Tompa, J. Am. Chem. Soc. 2015, 137, 13807–13817.
[15] R. K. Das, R. V. Pappu, Proc. Natl. Acad. Sci. 2013, 110, 13392–13397.
[16] K. J. Pacholarz, M. Porrini, R. A. Garlish, R. J. Burnley, R. J. Taylor, A. J. Henry, P. E.Barran, Angew. Chemie - Int. Ed. 2014, 53, 7765–7769.
[17] A. A. Russo, P. D. Jeffrey, N. P. Pavletich, Nat. Struct. Biol. 1996, 3, 696–700.
[18] S. R. Harvey, M. Porrini, A. Konijnenberg, D. J. Clarke, R. C. Tyler, P. R. R. Langridge-Smith, C. E. MacPhee, B. F. Volkman, P. E. Barran, J. Phys. Chem. B 2014, 118, 12348–12359.
[19] R. Sharma, Z. Raduly, M. Miskei, M. Fuxreiter, FEBS Lett. 2015, 589, 2533–2542.
[20] D. Stuchfield, P. E. Barran, Curr. Opin. Chem. Biol. 2018, 42, 177–185.
12
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