Correspondence/Rebuttal pubs.acs.org/biochemistry
The Quaternary Structure of the R‑State of Escherichia coli Aspartate Transcarbamoylase in Solution Is Different from That in the Crystal and Is Modified by Mg2+·ATP Binding L. Fetler† and P. Vachette*,‡ †
CERMES3, U. INSERM 988, CNRS UMR 8211, EHESS, Université Paris Descartes, BP 8, F-94801 Villejuif Cedex, France Institut de Biologie Intégrative de la Cellule, UMR 9198, Université Paris-Sud, F-91405 Orsay Cedex, France
‡
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few years ago, R. Kantrowitz and collaborators published in Biochemistry a series of three papers that constitute a remarkable contribution to the understanding of the mechanism of allosteric properties of Escherichia coli aspartate transcarbamoylase (ATCase).1−3,a The evidence they bring both functionally and structurally for the existence of a second, until then undetected nucleotide binding site for each regulatory subunit leads to the proposal of a molecular mechanism for often neglected observations such as the synergy between CTP and UTP and the role of Mg2+ ion that is always present in the bacterial cell. They report crystal structures of the ATCase−PALA complex (R-state) without a bound nucleotide, with ATP, and with Mg·ATP bound (one and two nucleotide molecules per chain respectively).2 The quaternary structures captured in the crystals are essentially identical as monitored by the distance between the top and bottom catalytic trimers that exhibits variations of only a few tenths of an angstrom. The importance of this work cannot be overestimated. In the last paper in the series,2 they write that “These results also indicate that the solution and crystal structures of ATCase are virtually identical” and further that “the RSOL·ATP and RSOL·ATP·Mg2+ structures of ATCase reported by Fetler and Vachette are incorrect” (they refer to ref 4). We first address their contentious claim that solution and crystal structures are virtually identical. Information about identity (or difference) between solution and crystal structures can be obtained from only a direct comparison between experimental data and intensities calculated from crystal structures. No experimental data are presented in ref 2, not even our published curves. We show three pairs of curves in Figure 1: dots with error bars are experimental data, while solid lines are intensity curves calculated from crystal structures after addition of a few missing N-terminal residues to the regulatory chains. Intensities were calculated using the program Crysol accounting for the contribution of hydration water.5 While the T-state experimental and calculated green curves (PDB entry 3d7s6) are very similar, in the case of the R-state, differences between calculated and experimental curves are observed regardless of the presence of Mg·ATP (PDB entry 1d09,7 blue without a nucleotide, and PDB entry 4kh0,2 orange with Mg·ATP). Intensities are plotted on a log scale, so that differences are actually quite large. Their statistical significance is provided by the corresponding χ2 values listed in Table 1. These differences prove that the solution structure of ATCase in the R-state is different from that within the crystal. This had © XXXX American Chemical Society
Figure 1. Comparison of experimental and calculated scattering curves of ATCase. Dots with error bars represent experimental data. Solid and dashed lines represent calculated curves from crystal structures: green solid line for the T-state (unliganded enzyme, PDB entry 3d7s), blue solid line for the R-state (PALA bound, PDB entry 1d09), orange solid line for PALA- and Mg·ATP-bound (PDB entry 4kh0), and orange dashed line for PDB entry 4kh0 from which Mg·ATP molecules were omitted in SAXS calculations.
already been reported in our work published almost 20 years ago8 and confirmed using an improved crystal structure (PDB entry 1d097) a few years later. Using rigid-body refinement of the structure, we proposed a model of the quaternary structure of the R-state accounting for our data, in which the distance between top and bottom catalytic trimers increases by 2.8 Å using the crystal R structure (PDB entry 1d09) as a reference with an associated rotation of each trimer in opposite directions.4 Finally, a recent study using molecular dynamics with explicit solvent is consistent with these conclusions: “ATCase in free simulations starting from RCRY spontaneously expands, coupled to relative rotation of the two trimers”. This suggests “that the crystal lattice stabilized a compact conformation”.9 Cockrell et al. also state that the contribution to scattering of Mg·ATP molecules accounts for the observed difference between the two R-state experimental scattering curves (Figure 1, blue and orange dots), without any conformational change. Received: February 21, 2017 Revised: May 15, 2017
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DOI: 10.1021/acs.biochem.7b00160 Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
Correspondence/Rebuttal
Table 1. χ2 Values for Comparisons of Calculated Intensities with Experimental Data
* 2
χ =
1 N−1
⎡ Iexp(qk) − cIcalc(qk) ⎤2 ∑k ⎢ ⎥⎦ , where c is a scaling factor and N the number of q values. σexp(qk) ⎣
Again, this statement needs to be substantiated by a quantitative comparison between experimental data of ATCase recorded with and without Mg·ATP (totally missing from ref 2) and calculated intensities from the corresponding crystal structures, with or without nucleotides in the computation of intensities. The calculated curve from their new crystal structure with Mg·ATP, 4kh0,2 is shown in Figure 1. The curve obtained from the same structure without nucleotides (Figure 1, dashed orange line) is practically identical to that obtained from the R-state structure with no bound nucleotide, 1d09 (Figure 1, blue line), as reported in Figure 8 of ref 2 and as expected from the similarity between the crystal quaternary structures. The difference between both calculated (orange solid and dashed) curves shows the contribution to scattering of bound nucleotides: while, experimentally, nucleotide binding causes a slight inward shift in the position of the minimum and a 25% increase in the intensity of the side maximum, the calculated curves show no shift and an ∼10% increase in side maximum intensity. To assess further the effect of bound nucleotides per se, we first used the rigid-body model for the R-state mentioned above and substituted the allosteric dimers with those from the 4kh0 crystal structure containing bound Mg·ATP. The scattering patterns of the resulting model calculated with and without Mg· ATP are colored red in Figure 2. The difference associated with nucleotides is significant but does not account entirely for that experimentally observed as confirmed by the χ2 value (Table 1). Similarly, using again rigid-body movement modeling, we obtained a good fit (χ2 = 3.2) to the experimental data recorded with Mg·ATP (dark gray solid line in Figure 2) by increasing the distance between the top and bottom trimers by 4.2 Å with respect to the 4kh0 crystal structure. This is 1.4 Å (50%) larger than the 2.8 Å increase observed for the R-state without a nucleotide. Finally, the scattering curve of this last model calculated without Mg·ATP molecules (dark gray dashed line in Figure 2) exhibits systematic differences with our experimental data recorded in the absence of nucleotides. Whatever the model used, the effect of bound nucleotides per se cannot account for the experimentally observed difference between scattering curves. In conclusion, the quaternary structure of the R-state of ATCase in solution is different from that in the crystal and the addition of Mg·ATP causes a significant conformational change that is probably associated with the change in the N-terminus of
Figure 2. Comparison of experimental and calculated scattering curves of ATCase with and without Mg·ATP. Close-up of the region of major change. Dots with error bars represent experimental data: blue for the R-state without a nucleotide and orange for the R-state with Mg·ATP. Lines represent scattering patterns calculated from models including (solid lines) or omitting (dashed lines) Mg·ATP: red lines for the rigid-body model of the solution R-state and the same model to which Mg·ATP molecules have been added and dark gray lines for the rigidbody model of the solution R-state with bound Mg·ATP and same model without nucleotides. See the text for details.
the regulatory chain that enters the allosteric site of the adjacent regulatory chain specifically observed in the presence of Mg·ATP.2 This can alter the structural dynamics of the molecule and explain the modification of the global quaternary structure observed in solution. Molecular dynamics calculations might shed some light on this point. In any event, we see here the limits of individual approaches in the study of complex molecular machines such as ATCase and the power of their combination into integrative, multiscale methods to elucidate their structural mechanism.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Telephone: +33 1 69 15 71 32. ORCID
P. Vachette: 0000-0002-2602-9704 B
DOI: 10.1021/acs.biochem.7b00160 Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
Correspondence/Rebuttal
Notes
The authors declare no competing financial interest.
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ABBREVIATIONS ATCase, aspartate transcarbamoylase; PALA, N-phosphonoacetyl-L-aspartate; PDB, Protein Data Bank; SAXS, small-angle X-ray scattering
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ADDITIONAL NOTE Because we discuss here some aspects of ref 2, we refer to it for all background information regarding ATCase. a
REFERENCES
(1) Cockrell, G. M., and Kantrowitz, E. R. (2012) Metal ion involvement in the allosteric mechanism of Escherichia coli aspartate transcarbamoylase. Biochemistry 51, 7128−7137. (2) Cockrell, G. M., Zheng, Y., Guo, W., Peterson, A. W., Truong, J. K., and Kantrowitz, E. R. (2013) New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase. Biochemistry 52, 8036−8047. (3) Peterson, A. W., Cockrell, G. M., and Kantrowitz, E. R. (2012) A second allosteric site in Escherichia coli aspartate transcarbamoylase. Biochemistry 51, 4776−4778. (4) Fetler, L., and Vachette, P. (2001) The allosteric activator MgATP modifies the quaternary structure of the R-state of Escherichia coli aspartate transcarbamylase without altering the TR equilibrium. J. Mol. Biol. 309, 817−832. (5) Svergun, D. I., Barberato, C., and Koch, M. H. J. (1995) CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768− 773. (6) Stieglitz, K. A., Xia, J., and Kantrowitz, E. R. (2009) The first high pH structure of Escherichia coli aspartate transcarbamoylase. Proteins: Struct., Funct., Genet. 74, 318−327. (7) Jin, L., Stec, B., Lipscomb, W. N., and Kantrowitz, E. R. (1999) Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue Nphosphonacetyl-L-aspartate at 2.1 A. Proteins: Struct., Funct., Genet. 37, 729−742. (8) Svergun, D. I., Barberato, C., Koch, M. H., Fetler, L., and Vachette, P. (1997) Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state. Proteins: Struct., Funct., Genet. 27, 110−117. (9) Chen, P. C., and Hub, J. S. (2015) Interpretation of solution x-ray scattering by explicit-solvent molecular dynamics. Biophys. J. 108, 2573−2584.
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DOI: 10.1021/acs.biochem.7b00160 Biochemistry XXXX, XXX, XXX−XXX
