Ion Mobility Mass Spectrometry of a Rotary ATPase Reveals ATP-induced Reduction in Conformational Flexibility
Rotary ATPases play fundamental roles in energy conversion, their catalytic rotation being associated with inter-domain fluctuations and heterogeneity of conformational states. Using ion mobility mass spectrometry (IM-MS) we compare the conformational dynamics of the intact ATPase from Thermus thermophilus (TtATPase) with its membrane and soluble subcomplexes. Our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. To provide a structural context for our experimental data we performed molecular dynamics (MD) simulations and observed conformational changes of the peripheral stalks reflecting their intrinsic flexibility. By isolating complexes at different phases of cell growth and manipulating nucleotides, metal ions and pH during isolation, we reveal differences that can be related to conformational changes in the Vo complex, triggered by ATP binding. Together these results implicate nucleotides in modulating flexibility of the stator components and uncover mechanistic detail underlying operation and regulation in the context of the holo-enzyme.
<!>TtAtPase (V1Vo) emerges from detergent micelles without major structural distortion<!>Intact V1Vo, V1 and Vo sub-domains display markedly different IM arrival time distributions<!>Reduced flexibility of Vo was attributed to ATP binding to the membrane subunit I<!>Conformational diversity of V1Vo is caused by \xe2\x80\x9cwobbling\xe2\x80\x9d of the catalytic head<!>Conformational broadening of V1\xe2\x80\x99 can be assigned to dynamics of the EG stalks<!>Discussion<!>Preparation of intact VoV1 complex and subcomplexes<!>Ion mobility mass spectrometry experiments<!>IM data analysis<!>MD simulations
<p>Rotary ATPases are highly efficient molecular nanomotors essential to biological energy metabolism. Classified into three subtypes F-, V- and A-type (also referred to as prokaryotic V-type), they share a conserved architecture comprising a water-soluble catalytic head and a membrane-embedded base, termed V1 and Vo in TtATPase, respectively1,2. TtATPase is the only ATPase in Thermus thermophilus and can operate in two directions towards ATP production or proton pumping depending on cellular requirements3,4. ATP synthesis/hydrolysis occurs in V1 and is coupled to H+/Na+ translocation in the membranous Vo via a rotational motion of the subunits D, F, C anchored to a membrane embedded ring (the rotor). The remaining subunits constitute the stator including the AB hexamer and peripheral stalks in V1 that connect to subunit I in Vo. A wealth of structural information is available for individual components of V1, whereas the membranous Vo and intact V1Vo complex resist high-resolution structural elucidation1,5. The difficulty arises in part from the flexibility required for the synergetic cooperation of components from both soluble and membrane domains, resulting in heterogeneity of conformational states along the time axis of rotary catalysis. This combined with the amphipathic nature and dynamic disassembly6,7 of intact complexes presents significant challenges for traditional structural biology approaches.</p><p>Recent discovery of conditions for the introduction of membrane-embedded assemblies into the gas phase and its coupling with ion mobility mass spectrometry (IM-MS) has enabled conformation and dynamics of membrane proteins to be assessed. The method exploits the protective effect of detergent micelles during transfer and gas phase excitation to release complexes from detergent encapsulation8,9 enabling native-like structures to be retained10,11. This implies that collision energy is directed primarily towards disruption of detergent assemblies without perturbing the structures of membrane complexes.</p><p>IM separation is based on the ability of ions to traverse a gas-filled chamber under the influence of an electric field12. Ions of different size and shape experience different resistance along their migration colliding with inert gas molecules13,14,15; their arrival time distributions (ATD) are recorded and converted to orientationally averaged collision cross sections (CCS)16,17. Experimental CCSs can be used as restraints to guide structural modelling of protein complexes18,19. ATD peaks are generally Gaussian but for complexes in which unfolding occurs, asymmetric peaks, skewed towards slower arrival times, are observed17,20. Structural dynamics and heterogeneity can be inferred from the distribution of mobilities; ions with multiple conformations resulting in broad Gaussian shaped ATD peaks10,15.</p><p>Recently we demonstrated that two prokaryotic V-type ATPases, encapsulated in detergent micelles, could be projected into the vacuum of a mass spectrometer. Release of "naked" ions by collisional excitation enabled us to explore their subunit interactions, lipid binding properties and the structural consequence of nucleotide binding21. Here we extend our study using IM-MS to characterise the 3D topology of TtATPase. We show that native-like structure can be preserved in vacuum and contrast variation in CCS of the intact complex with different subcomplexes enabling us to assign dynamics to the peripheral stalks, in accord with MD simulations. By exploring in vivo and in vitro conditions we reveal conformational changes in Vo triggered by ATP binding. Collectively these results relate nucleotide binding to stability and link conformational flexibility to cellular ATP/ADP levels.</p><!><p>To assess the overall topology in the absence of bulk membrane lipids or detergents we acquired IMMS spectra for intact TtATPase (Fig. 1). Under carefully controlled conditions three charge state series were recorded and assigned to intact V1Vo, disassembled V1 (A3B3DF) and membranous Vo (ICL12). Reduction in charge was observed for species containing transmembrane regions (V1Vo and Vo, Supplementary Table S1), consistent with reduced accessible surfaces due to the presence of detergent micelles22,23. ATDs recorded for all three species are well-defined Gaussians implying that they emerge from detergent micelles without major structural distortion.</p><p>To correlate IM data with solution phase structures, centroids were extracted from the IM peaks and calibrated against a CCS database of proteins and protein complexes24. Experimental CCS were compared with theoretical values calculated for structures assembled by docking, homology and coarse-grained modelling, guided by the EM density map (Supplementary Methods). Good agreement was observed for all three species V1, Vo and intact V1Vo (Table 1, all within 1.0%) implying that the conformations of these complexes in the gas phase do not deviate significantly from their anticipated structures.</p><!><p>We noticed that the three species V1, Vo and V1Vo within the same IM-MS spectrum display well-resolved Gaussian peaks but have markedly different widths (Fig. 1 (i-iii)). In comparison to the intact V1Vo, ATDs recorded for V1 (A3B3DF) are much narrower while for Vo (ICL12) much broader ATDs are observed. We computed average arrival time resolution (Rt), expressed as t/Δt, which is affected by experimental conditions and dictated primarily by diffusion of ions through the IM chamber25,26. It is expected that theoretical Rt values for V1, Vo and V1Vo should remain steady (estimated 12% lower for Vo and 5% higher for VoV1
cf. V1, Supplementary Methods). Rt values measured experimentally however span a much wider range (70% and 44% lower for Vo and VoV1 respectively than V1, Table 1).</p><p>Since Rt is both instrument and ion-dependent we converted Rt values to RΩ (centroid CCS normalised to width at half-height). IM peaks assigned to V1 are sharp with an average RΩ of 34.6 corresponding to a CCS variation of 2.9%, close to the limiting resolution of the instrumentation employed25,27, implying that V1 exists in one principal conformation similar to the X-ray structure28. By contrast Vo has a much lower RΩ (10.8) consistent with its CCS span being three-fold greater than V1 (9.2% vs. 2.9%, Table 1). This broadening likely reflects heterogeneity characteristic of an ensemble of conformations populated by Vo.</p><!><p>To investigate the origin of this conformational heterogeneity we explored a series of in vivo and in vitro conditions, monitoring their effects on the structural dynamics of Vo. Conditions included: (i) harvesting cells at nutrient-abundant vs. nutrient-limited status; (ii) pH manipulation (6.8 or 8.2); (iii) use of chelating EDTA to sequester metal ions; and (iv) ATP or AMPPNP supplement to purification buffers, leading to 12 different purifications (Table 3). Results showed that cellular nutrition status, pH, metal ions or AMPPNP had no appreciable effect on the CCS of Vo (Table 2). By contrast, ATD peaks recorded for Vo in all preparations supplemented with 1mM ATP shifted to shorter drift times, indicative of an average structural compaction of 146 Å2 (Fig. 2a, left panel). This CCS reduction was observed for all ATP-supplemented preparations with reproducibility (Fig. 2b & 2c), and was invariably accompanied by a narrower mobility distribution (higher RΩ, Table 2). Our IM data therefore point to a compact, defined structure of Vo induced by ATP binding.</p><p>To identify the cause of this structural compaction we performed in-solution disassembly of Vo (ICL12) generating CL12 from loss of subunit I. CL12 exhibited much narrower CCS distributions than ICL12, with a CCS close to that calculated from the modelled structure (Fig. 2a, right panel) allowing us to assign conformational heterogeneity to subunit I. Moreover lack of ATP-associated changes of IM profiles for CL12 further pinpointed subunit I as being responsible for ATP-induced compaction and reduction in dynamics of Vo. Therefore in preparations lacking ATP the broad ATD peaks of Vo are associated with dynamics of subunit I, while ATP not only reduces the CCS of Vo by ca. 146 Å2 but also narrows the distribution from 9 to 6% (Table 2).</p><p>To account for the CCS difference of Vo in the presence and absence of ATP, we built a structural model of Vo reproducing its conformation within intact TtATPase (Supplementary Methods), in which the soluble portion of I (Isol) runs parallel to the membrane plane and forms a ~90° angle with its membrane domain (Imem) (Fig. 2 (vi)). Theoretical CCS estimated for this model agrees well with measured value of Vo in the presence of ATP. In its absence the observed CCS of Vo increases implying deviation of Isol from its original conformation. To estimate the conformational flexibility of subunit I we performed normal mode analysis (NMA)29 computing the low frequency modes of Isol, employing the crystal structure of the yeast analogue of Isol (subunit C, PDB code: 1U7L)30. NMA established elasticity in the long α-helical region in the middle part of the structure. This allowed the distal end of Isol to flex upward with an offset of 10 Å and a 15° angle, resulting in an alternative conformation that we referred to as Isol' (Supplementary Fig. S2). Incorporating this elasticity into the model of Vo led to a predicted CCS difference of 137 Å2 (Fig. 2, (iii) vs. (vi)), in line with the CCS deviation observed experimentally (Table 2, 146 Å2). Interestingly Isol' produced by NMA mirrors a second structure of subunit C solved at 2.9 Å30.</p><p>To correlate CCS distributions of Vo with conformational dynamics we generated a series of structural models for Vo incorporating Isol or Isol', varying the angle with Imem in 5° increments, from 80° up to 150°. Theoretical CCSs were calculated and compared with experimental values, enabling us to estimate an angular freedom for Isol in the presence of ATP of ca. 30° (80° to 110°, Supplementary Table 3). By contrast absence of ATP caused Isol' to have enhanced angular freedom ca. 60° (80° to 140°) (Fig. 2d & 2e). Taken together these results highlight a nucleotide-induced change in the dynamics of subunit I in the isolated Vo complex.</p><!><p>This dynamic motion of subunit I, with and without ATP, would likely be restrained within the intact V1Vo complex by interaction with the peripheral EG stalks31,32. Interestingly ATD peaks recorded for intact VoV1 are less broad than those of Vo (RΩ 18.5 vs. 10.8 respectively, Table 1), suggesting a smaller conformational space occupied by V1Vo. Since both complexes contain subunit I this implies that its conformational dynamics observed in ICL12 are restrained in the intact assembly. However, even with the movement of I constrained it is not possible to achieve similar IM resolution for VoV1 to that of V1 (RΩ 18.5 vs. 34.6) implying that residual dynamics remains for VoV1.</p><p>We considered the characteristic "wobbling"33,34 of the catalytic head, consistent with inclination of V1 relative to Vo, to estimate the CCS variation for the intact complex. Taking into account rotation of the central stalk, and flexing of both peripheral stalks adopting alternative conformations during rotation, a theoretical CCS difference of 3.7% was anticipated (Supplementary Table S4). A variation of 5.4 ± 0.9% was measured by IM (Table 1, see below, Fig. 4a). This conformational broadening, when the motion of subunit I is restrained, mirrors an ensemble of coexisting structures of the intact TtATPase, possibly trapped at different stages along the dynamic process of rotary catalysis.</p><p>To support this proposal we probed the dynamic nature of the intact complex using an antibody (dAb) that recognises the EG stalks with high specificity35. MS spectra recorded for V1Vo following incubation with a large excess of dAb reveal sub-stoichiometric binding, implicating different affinities of the two EG stalks in binding dAb (Supplementary Methods). We interpret this as evidence for a conformational divergence associated with the EG stalks, in accord with their role in accommodating the wobbling rotation of the asymmetrical complex.</p><!><p>Next we turned our attention to the isolated V1 subcomplex, formed by loss of both peripheral stalks as well as the membrane Vo complex. Sharp ATDs were observed consistent with minimal variation in CCS for V1 (RΩ = 34.6, Table 1). This illustrates uniformity of conformations, in keeping with the 3-fold symmetry of V1 around the central axis perpendicular to the A3B3 plane (Fig. 1(ii)); structures of V1 after rotation of the central stalk by 0, 120 and 240° are superimposable. Conformational fluctuation associated with intermediate states however cannot be ruled out36 but likely occurs on a minor scale since they are not detected with our IM instrumentation25,37. We conclude that the soluble head, in the absence of the peripheral stalks, exhibits the least conformational heterogeneity.</p><p>Disassembly of the intact complex in solution leads to the V1' assembly with two peripheral stalks attached. IM data of V1' give an experimental CCS of 172 nm2 (Fig. 1 right), cf. 173 nm2 calculated for V1' assembled by docking the peripheral stalks onto V1 (Table 1, SI). Overlapping peaks of low intensity around 9,000 m/z arise primarily from loss of both stalks as well as one B subunit (A3B2DF, Fig. 1 (v), Supplementary Table S1). Comparing the IM peak widths recorded for V1' with those of A3B2DF, generated in the same solution and subject to the same MS conditions (Table 1), clearly demonstrates a conformational diversity for V1', not present in A3B2DF, assigned to the binding of the peripheral stalks.</p><p>The peripheral EG stalk features a coiled coil tail extruding 120 Å from the main structure31 (Fig. 1 (iv)). The close correlation of the CCS measurement with the native topology of V1', as well as significant structural heterogeneity demonstrated for the assembly (Table 1) is in contrast to a previous IM-MS study which revealed collapse of intrinsically disordered loops onto a folded "core"38. To explore the gas phase behaviour of V1' we carried out in vacuo MD simulations (Fig. 3). Interestingly no collapse of peripheral stalks was observed during the course of simulations (1 nanosecond). On the contrary, MD simulations illustrated remarkable flexibility of distal ends of the stalks, while the globular domains, anchored to the catalytic head (V1), remained essentially unaltered.</p><p>It is interesting to consider flexibility of V1' in the light of distinct structures of the T. thermuphilus peripheral stalks reported recently31,33. Models of V1' incorporating combinations of these structures and their inter-conversion, give a calculated CCS difference of up to 1026 Å2 (Fig. 3c & S3). Selecting two V1' models that yield the largest CCS difference and comparing their MD simulations (Fig. 3a & b, movie S1 & 2) initially for both models we observe conformational fluctuations, which start to stabilise after ca. 0.2 ns. Comparing CCS changes over the second 0.5 ns period of both simulations we obtain a theoretical value of 4.5%.</p><p>To compare this theoretical variation with the value established experimentally for V1' (7.0±1.0%, Table 1), we considered all four species with conformational heterogeneity investigated here (Vo, V1' V1 and A3B2DF) and plotted their theoretical CCS variations against experimental values (Fig. 4a). An excellent linear correlation was found for two datasets with a positive y-interception of >2.0%, close to the limit set by the current IM approach25. The close correlation therefore verifies our conclusion of increased conformational flexibility assigned to the peripheral stalks during rotary catalysis, a mechanism that accommodates "wobbling" of the catalytic head.</p><!><p>Here we report an in-depth IM-MS investigation of the intact TtATPase and demonstrate that it is possible to retain the structural integrity of this rotary enzyme and its subcomplexes, in the absence of bulk solvent and detergent. We show that the broad ATD pattern for Vo is attributed to flexibility of subunit I, and demonstrate compaction and reduction in dynamics in the presence of ATP. We also provide experimental evidence for flexibility of the EG stalks, closely reflected by MD simulations. Incorporation of this flexibility enabled us to assess the scope of conformational change for the intact ATPase during rotary catalysis, and more importantly, to explore a nucleotide regulatory mechanism of the complex responding to unfavourable cellular conditions, similar to the reversible disassembly established in eukaryotic V-ATPases (Fig. 4b).</p><p>Although the catalytic and regulatory roles of nucleotides in the hydrophilic V1-headpiece have been established28,36, their binding in the membranous Vo are less well-understood. Nucleotide-binding capabilities however have been reported for the C subunits of S. cerevisiae and A. thaliana V1Vo39, functional analogues of Isol in TtATPase6. Accordingly lack of ATP was proposed to promote disassembly of isolated Vo domain of TtATPase21. Prompted by these initial observations here we carried out a systematic study assessing the role of a variety of factors on the conformational dynamics of isolated Vo. The study uncovered a role for ATP in stabilising the native conformation of Isol restricting its mobility; depletion of ATP was shown not only to trigger a conformational change in Isol converting to Isol', but also to increase its freedom of movement from an angular range of 30° in the presence of ATP, to 50° in its absence.</p><p>Subunit I is the least understood yet evolutionarily conserved component across the ATPase family, for which no structural information is available. As the sole amphipathatic protein of TtATPase its Isol and Imem domains, connected by an intermediate 'hinge' region21,39, tether the EG stalks and constitute the proton channel across the membrane, respectively. Elasticity of the hinge region is obligatory to enable mobility of the periplasmic components without disrupting integrity of the transmembrane channel. Considering nucleotide-induced changes of Vo when ATP is supplemented, fluctuation of Isol may well reflect elasticity of subunit I enabling dynamic movement of the catalytic head in a wobbling ATPase. Conversely when ATP is depleted our observation points to a mechanism of how cellular ATP levels are sensed and regulate the intact enzyme: in lack of ATP, ADP (or other nucleotides) likely competes for binding in the hinge domain of I and induces a conformational change converting Isol to Isol'. Optimal interaction of subunit I formed with the EG stalks is therefore compromised resulting in a strained complex of reduced stability, prone to disassemble into Vo and V1. Once separated, increased mobility of Isol in Vo further destabilises interaction of Imem with the rotor ring (CL12) leading to dissociation of subunit I. The mechanism is underpinned by minimal contact observed between I and CL12 ring by EM40, and is postulated as a means for silencing the proton translocating Vo detached from the intact complex21.</p><p>We speculate that ATP could execute its effect through: (i) competing at higher concentration for the nucleotide binding site in the hinge region of I which preferentially binds ADP21,39, hence preventing a conformational change and relaxation associated with ADP binding; (ii) transient binding to a different site enhancing conformational stability of Vo. Either way our observations suggest a generality of nucleotide-induced conformational changes in the stator connecting the cytoplasmic V1 and membranous Vo domains. Cellular ATP concentration is typically 1–10 mM41; the available ATP pool is expected to be in the sub-millimolar range given its association with the many molecular machineries involved in a multitude of energy-dependent processes42. Our results therefore bear physiological implications for the in vivo control mechanism via ATP/ADP ratios by showing a stabilisation by 1mM ATP while an ADP concentration of > 50μM is sufficient for relaxation of Vo21.</p><p>A characteristic feature of eukaryotic V-type ATPases is the regulatory reversible disassembly of V1 and Vo domains5. By demonstrating the dynamic properties of the EG stalks and subunit I in isolated V1 and Vo of TtATPase respectively, our IM-MS data point to a critical role of the stator components in maintaining stability of intact VoV1. Peripheral stator components impart characteristics unique to V-ATPases; they must balance the stability essential for productive catalysis with the dynamic instability involved in regulation. Our results therefore raise the intriguing possibility of a reversible dissociation mechanism in analogy to eukaryotic V-type ATPases, likely mediated by the subunit I and peripheral stalks43, in response to cellular ATP/ADP levels for the bacterium Thermus thermophilus. Binding of ATP favours the active conformation of I in V1Vo maintaining an efficient ion pump. Conversely when ATP is depleted the complex reverts towards ATP synthesis; the integrity of which is maintained through a mechanism involving transmembrane proton motive force (PMF). Lack of both ATP and PMF (mimicking a nutrient-deprived state) leads to conformational changes associated with increased dynamics induced by ADP binding in Isol perturbing its optimal binding with the EG stalks, serving to initiate the cooperative "unloading" of V1 from Vo. In this regard it is of interest to consider an early result which showed binding of S. cerevisiae C subunit (Isol analogue) to the M. Sexta V1 lacking endogeneous subunit C (V1-C) significantly enhanced its ATP hydrolysis44, supporting the notion that restriction of the peripheral stalks is desirable for optimal ATPase activity.</p><p>High-resolution structural biology tools have generated an ever-increasing database of static structures; their dynamic conformations however are among the most difficult to assess despite the fact that these often underpin the function and regulation of macromolecular machineries. Here we have shown how distribution of ATD can be used to assess rotations and conformational fluctuations in membrane-embedded rotary motors. With the aid of structural modeling and in vacuo MD simulation we relate these distributions to conformational flexibility, essential for the cooperativity and regulation of the dynamic nanomotor. Plotting theoretical values against experimental data enables us to isolate conformational heterogeneity from a background of factors contributing to IM distributions. Overall our results not only allow elucidation of nucleotide regulated flexibility for the TtATPase, but also highlight the potential of IM-MS to probe subtle conformational changes within membrane-embedded assemblies that often elude high-resolution structural characterisation due to averaging across populations of interconverting states.</p><!><p>Intact VoV1 was purified from Thermus thermophilus strain HB8 as previously described32,35. Thermus thermophilus cells were grown at 65°C (150 rpm) to stationary or log phase, and purified with supplements indicated in Table 3 (see SI for details). Purified VoV1 were buffer exchanged into 200mM AmAc and diluted to 1.1 mg/mL prior to IM-MS experiments. The CL12 subcomplex was generated by decreasing the pH21, and disassembly to V1' subcomplex was achieved via a further dilution (1:10) bringing the DDM concentration well below its critical micelle concentration (CMC) with IM-MS spectra immediately recorded.</p><!><p>All IM-MS spectra were recorded on hybrid quadrupole (Q)-IM-ToF MS instruments known as Synapt27 or Synapt G237 HDMS systems incorporating travelling-wave ion guide for IM separation (Waters, UK). Both instruments are modified for high mass45 and use nitrogen as mobility gas with the trap and transfer regions filled with argon except for measurement of the intact VoV1 sulphur hexafluoride (SF6) was used. Instrument conditions were optimized for high m/z ions with acceleration kept minimal to avoid thermal heating of ions. Specifically ions were injected into the mobility cell at a 100 μs pulse with a voltage of 18V. The pressures of Synapt were maintained at 4.33e−1 and 3.42e−2 mbar in the mobility and trap/transfer regions, respectively. Triplicate measurements employing different combinations of wave height (WH) and wave velocity (WV) are listed (Supplementary Table S2). The Synapt G2 system was operated at higher pressures of 3.25 and 3.95e−2 mbar for mobility and trap/transfer regions, respectively, which are separated by a 'helium gate' pressurized at 1.43 bar. Triplicate experiments were carried out with a fixed WH of 40V and varied WV at 550, 600 and 640ms−1 (Supplementary Table S2). Measurement of the disassembled V1' was taken on the Synapt operated at 3.96e−1 and 5.93e−2 mbar for the mobility and trap/transfer regions, respectively. Mobility separations were performed with a fixed WV of 300ms−1 and varied WH at 8, 10 and 11V. CCS Measurement for the Vo (ICL12) and CL12 subcomplexes was carried out on Synapt G2 maintaining 1.49 and 3.08e−2 mbar for mobility and trap/transfer regions, respectively, and helium gate pressurized at 9.66e−1 bar. Mobility separation was performed with a WH of 31V and WV of 800ms−1. The Measurements were both replicated four times under identical instrumental conditions.</p><!><p>Individual IM arrival time distributions (ATD) were extracted and converted to collision cross section (CCS) distributions using a calibration procedure described elsewhere17, based on a set of proteins and protein complexes used as IM standards encompassing a wide mass range24. For each extracted ATD, arrival time resolution (Rt) was measured as the peak centroid divided by its width at 50% height (t/Δt). Similarly cross section resolution (RΩ) was calculated as the centroid CCS value divided by the span of the CCS distribution at 50% height (Ω/ΔΩ). The CCS variations are the average half-height spans of the CCS distributions normalized to their respective centroid CCSs. The Rt, RΩ, experimental CCS and CCS variations reported in Table 1 are average values across each charge state series.</p><!><p>The simulations were performed in double floating-point precision with GROMACS 4.5.3 using the OPLS-AA/L force field20,46. The procedure started with a steepest descent energy minimization followed by 1 ns vacuum equilibration with randomly generated initial velocities. Neither periodicity nor cut-offs were employed in the calculations. Energy conservation was achieved using a 1 fs integration step, and constraining bonds to hydrogen with the LINCS algorithm47. A dielectric constant of ε = 2ε0 (where ε0 is the dielectric permittivity of free space) was used to describe the gas-phase basicity in our calculations48. The simulations were run at constant temperature of 300 K. To assign charged residues we employed an in-house developed search algorithm which evenly distributed charges over basic residues <5 Å from the surface20. Two alternative configurations were prepared for each complex, and three trajectories were analysed with respect to their RMSD from the initial structure. An RMSD-clustering algorithm was used to identify the most populated conformations at different stages in each trajectory, and CCSs were calculated for structures every 20 picoseconds (see SI for details).</p>