Preliminary X-ray Diffraction Analysis of the Membrane Protein styMdtM from Salmonella Typhi
DOI:
https://doi.org/10.53560/PPASB(62-4)1125Keywords:
Major Facilitator, Efflux Transporter, styMdtM, STY4874, Membrane Protein, Salmonella TyphiAbstract
Structural studies of proteins provide a comprehensive understanding of their functions, and form the basis for designing drugs that can modulate their activity. However, the structural characterization of membrane proteins is often challenging due to their hydrophobic nature. This article highlights key structural and functional aspects of a representative membrane protein, STY4874 – also known as styMdtM, which is an efflux transporter encoded by the genome of Salmonella Typhi, the causative agent of typhoid fever in humans. Our structural studies have, so far, been focused on obtaining diffraction-quality crystals of the protein. For this purpose, crystallization trials have been performed using both the wild-type and mutant forms of the protein, in the presence or absence of ligands (substrates or inhibitors). Crystals of the wild-type styMdtM and its thermally stable mutants diffracted anisotropically to resolutions between 4.5 and 8 Å, rendering the collected data not useful for structure solution. On the other hand, crystals of a 13-amino acid deletion mutant isotropically diffracted to 10 Å, fueling our interest in engineering truncated styMdtM mutants for obtaining crystals suitable for diffraction to high resolutions for structure determination. Here, we discuss preliminary findings and key lessons learned from our studies, spanning over a decade.
References
J. Garcia-Nafria, and C.G. Tate. Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development. Annual Review of Pharmacology & Toxicology 60: 51-71 (2020).
2. J.A. Purslow, B. Khatiwada, M.J. Bayro, and V. Venditti. NMR Methods for Structural Characterization of Protein-Protein Complexes. Fronteiers in Molecular Bioscience 7: 9 (2020).
3. H. Xie, Y. Zhao, W. Zhao, Y. Chen, M. Liu, and J. Yang. Solid-state NMR structure determination of a membrane protein in E. coli cellular inner membrane. Scientific Advancement 9(44): eadh4168 (2023).
4. S. Narasimhan, C. Pinto, A. Lucini Paioni, J. van der Zwan, G.E. Folkers, and M. Baldus. Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy. Nature Protocol 16(2): 893-918 (2021).
5. F. Li, P.F. Egea, A.J. Vecchio, I. Asial, M. Gupta, J. Paulino, R. Bajaj, M.S. Dickinson, S. Ferguson-Miller, B.C. Monk, and R.M. Stroud. Highlighting membrane protein structure and function: A celebration of the Protein Data Bank. Journal of Biological Chemistry 296: 100557 (2021).
6. Q. Ain, M. Tahir, A. Sadaqat, A. Ayub, A.B. Awan, M. Wajid, A. Ali, M. Iqbal, A. Haque, and Y. Sarwar. First Detection of Extensively Drug-Resistant Salmonella Typhi Isolates Harboring VIM and GES Genes for Carbapenem Resistance from Faisalabad, Pakistan. Microbial Drug Resistance 28(12): 1087-1098 (2022).
7. J. Akram, A.S. Khan, H.A. Khan, S.A. Gilani, S.J. Akram, F.J. Ahmad, and R. Mehboob. Extensively Drug-Resistant (XDR) Typhoid: Evolution, Prevention, and Its Management. Biomedical Research International 2020: 6432580 (2020).
8. A. Shaheen, A. Tariq, F. Ismat, H. Naveed, R. De Zorzi, M. Iqbal, P. Storici, O. Mirza, T. Walz, and M. Rahman. Identification of additional mechanistically important residues in the multidrug transporter styMdtM of Salmonella Typhi. Journal of Biomolecular Structure & Dynamics 42(21): 11641-11650 (2024).
9. H.H. Wu, J. Symersky, and M. Lu. Structure of an engineered multidrug transporter MdfA reveals the molecular basis for substrate recognition. Communication Biology 2: 210 (2019).
10. H.H. Wu, J. Symersky, and M. Lu. Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily. Scientific Report 10(1): 3949 (2020).
11. E.H. Yardeni, T. Bahrenberg, R.A. Stein, S. Mishra, E. Zomot, B. Graham, K.L. Tuck, T. Huber, E. Bibi, H.S. McHaourab, and D. Goldfarb. Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels. Scientific Report 9(1): 12528 (2019).
12. E.H. Yardeni, S. Mishra, and R.A. Stein, E. Bibi, H.S. McHaourab. The Multidrug Transporter MdfA Deviates from the Canonical Model of Alternating Access of MFS Transporters. Journal of Molecular Biology 432(20): 5665-5680 (2020).
13. K.O. Alegre, S. Paul, P. Labarbuta, and C.J. Law. Insight into determinants of substrate binding and transport in a multidrug efflux protein. Scientific Report 6: 22833 (2016).
14. A. Shaheen, F. Ismat, M. Iqbal, A. Haque, R. De Zorzi, O. Mirza, T. Walz, and M. Rahman. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi. Journal of Infection & Chemotherapy 21(5): 357-362 (2015).
15. A. Shaheen, F. Ismat, M. Iqbal, A. Haque, Z. Ul-Haq, O. Mirza, R. De Zorzi, T. Walz, and M. Rahman. Characterization of the multidrug efflux transporter styMdtM from Salmonella enterica serovar Typhi. Proteins 89(9): 1193-1204 (2021).
16. A. Tariq, M. Sana, A. Shaheen, F. Ismat, S. Mahboob, W. Rauf, O. Mirza, M. Iqbal, and M. Rahman. Restraining the multidrug efflux transporter STY4874 of Salmonella Typhi by reserpine and plant extracts. Letters in Applied Microbiology 69(3): 161-167 (2019).
17. S. Iwata (Ed.). Methods and results in crystallization of membrane proteins. International University Line La Jolla, California (2003).
18. M.B. Khan, G. Sponder, B. Sjoblom, S. Svidova, R.J. Schweyen, O. Carugo, and K. Djinovic-Carugo. Structural and functional characterization of the N-terminal domain of the yeast Mg2+ channel Mrs2. Acta. Crystallographica, Section D: Biological Crystallography 69(Pt 9): 1653-1664 (2013).
19. A. McPherson. A comparison of salts for the crystallization of macromolecules. Protein Science 10(2): 418-422 (2001).
20. A. Shaheen. Characterization of efflux pumps conferring multidrug resistance in Salmonella enterica serovar Typhi. Ph.D. Thesis. Pakistan Institute of Engineering and Applied Sciences, Islamabad, Pakistan (2016).
21. M. Varadi, S. Anyango, M. Deshpande, S. Nair, C. Natassia, G. Yordanova, D. Yuan, O. Stroe, G. Wood, A. Laydon, A. Zidek, T. Green, K. Tunyasuvunakool, S. Petersen, J. Jumper, E. Clancy, R. Green, A. Vora, M. Lutfi, M. Figurnov, A. Cowie, N. Hobbs, P. Kohli, G. Kleywegt, E. Birney, D. Hassabis, and S. Velankar. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research 50(D1): D439-D444 (2022).
22. E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, and T.E. Ferrin. UCSF Chimera-a visualization system for exploratory research and analysis. Journal of Computational Chemistry 25(13): 1605-1612 (2004).
23. R.M. Bill, P.J. Henderson, S. Iwata, E.R. Kunji, H. Michel, R. Neutze, S. Newstead, B. Poolman, C.G. Tate, and H. Vogel. Overcoming barriers to membrane protein structure determination. Nature Biotechnology 29(4): 335-340 (2011).
24. S.R. Holdsworth and C.J. Law. Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM. Biochimie 94(6): 1334-1346 (2012).
25. S.R. Holdsworth and C.J. Law. The major facilitator superfamily transporter MdtM contributes to the intrinsic resistance of Escherichia coli to quaternary ammonium compounds. Journal of Antimicrobial Chemotherapy 68(4): 831-839 (2013).
26. S.R. Holdsworth and C.J. Law. Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli. BMC Microbiology 13: 113 (2013).
27. J. Adler and E. Bibi. Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain. Journal of Bacteriology 184(12): 3313-3320 (2002).
28. J. Adler and E. Bibi. Determinants of substrate recognition by the Escherichia coli multidrug transporter MdfA identified on both sides of the membrane. Journal of Biological Chemistry 279(10): 8957-8965 (2004).
29. J. Adler and E. Bibi. Promiscuity in the geometry of electrostatic interactions between the Escherichia coli multidrug resistance transporter MdfA and cationic substrates. Journal of Biological Chemistry 280(4): 2721-2729 (2005).
30. J. Adler, O. Lewinson, and E. Bibi. Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA. Biochemistry 43(2): 518-525 (2004).
31. R. Edgar and E. Bibi. A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA. EMBO Journal 18(4): 822-832 (1999).
32. N. Sigal, E. Vardy, S. Molshanski-Mor, A. Eitan, Y. Pilpel, S. Schuldiner, and E. Bibi. 3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge. Biochemistry 44(45): 14870-14880 (2005).
33. E. Zomot, E.H. Yardeni, A.V. Vargiu, H.K. Tam, G. Malloci, V.K. Ramaswamy, M. Perach, P. Ruggerone, K.M. Pos, and E. Bibi. A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter. Journal of Molecular Biology 430(9): 1368-1385 (2018).
34. M. Levantino, B.A. Yorke, D.C. Monteiro, M. Cammarata, and A.R. Pearson. Using synchrotrons and XFELs for time-resolved X-ray crystallography and solution scattering experiments on biomolecules. Current Opinion in Structural Biology 35: 41-48 (2015).
35. J.D. Gunton, A. Shiryayev, and D.L. Pagan (Eds.). Protein condensation: kinetic pathways to crystallization and disease. Cambridge University Press (2007).
36. S.T. Yau, D.N. Petsev, B.R. Thomas, and P.G. Vekilov. Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals. Journal of Molecular Biology 303(5): 667-678 (2000).
37. D.N. Petsev, B.R. Thomas, S.T. Yau, D. Tsekova, C. Nanev, W.W. Wilson, and P.G. Vekilov. Temperature-independent solubility and interactions between apoferritin monomers and dimers in solution. Journal of Crystal Growth 232(1): 21-29 (2001).
38. O. Gliko, N. Neumaier, W. Pan, I. Haase, M. Fischer, A. Bacher, S. Weinkauf, and P.G. Vekilov. A metastable prerequisite for the growth of lumazine synthase crystals. Journal of the American Chemical Society 127(10): 3433-3438 (2005).
39. P.G. Vekilov, A. Feeling-Taylor, S.T. Yau, and D. Petsev. Solvent entropy contribution to the free energy of protein crystallization. Acta Crystallography Section D: Biological Crystallography 58(10): 1611-1616 (2002).
40. Z.S. Derewenda and P.G. Vekilov. Entropy and surface engineering in protein crystallization. Acta Crystallography Section D: Biological Crystallography 62(1): 116-124 (2006).
41. P.G. Vekilov. Solvent entropy effects in the formation of protein solid phases. Methods in Enzymology 368: 84-105 (2003).
42. Z.S. Derewenda. Application of protein engineering to enhance crystallizability and improve crystal properties. Acta Crystallography Section D: Biological Crystallography 66(5): 604-615 (2010).
43. M.C. Botfield and T.H. Wilson. Carboxyl-terminal truncations of the melibiose carrier of Escherichia coli. Journal of Biological Chemistry 264(20): 11643-11648 (1989).
44. P.D. Roepe, R.I. Zbar, H.K. Sarkar, and H.R. Kaback. A five-residue sequence near the carboxyl terminus of the polytopic membrane protein lac permease is required for stability within the membrane. Proceedings of National Academy of Science USA. 86(11): 3992-3996 (1989).
45. K. Sato, M.H. Sato, A. Yamaguchi, and M. Yoshida. Tetracycline/H+ antiporter was degraded rapidly in Escherichia coli cells when truncated at last transmembrane helix and this degradation was protected by overproduced GroEL/ES. Biochemical and Biophysical Research Communication 202(1): 258-264 (1994).
46. J. Jumper, R. Evans, A. Pritzel, T. Green, M. Figurnov, O. Ronneberger, K. Tunyasuvunakool, R. Bates, A. Zidek, A. Potapenko, A. Bridgland, C. Meyer, S.A.A. Kohl, A.J. Ballard, A. Cowie, B. Romera-Paredes, S. Nikolov, R. Jain, J. Adler, T. Back, S. Petersen, D. Reiman, E. Clancy, M. Zielinski, M. Steinegger, M. Pacholska, T. Berghammer, S. Bodenstein, D. Silver, O. Vinyals, A.W. Senior, K. Kavukcuoglu, P. Kohli, and D. Hassabis. Highly accurate protein structure prediction with AlphaFold. Nature 596(7873): 583-589 (2021).
47. D. Drew, R.A. North, K. Nagarathinam, and M. Tanabe. Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS). Chemical Reviews 121(9): 5289-5335 (2021).
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Upon acceptance of an article, its copyright will be assigned to the Pakistan Academy of Sciences.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Creative Commons Attribution (CC BY). Allows users to: copy the article and distribute; abstracts, create extracts, and other revised versions, adaptations or derivative works of or from an article (such as a translation); include in a collective work (such as an anthology); and text or data mine the article. These uses are permitted even for commercial purposes, provided the user: includes a link to the license; indicates if changes were made; gives appropriate credit to the author(s) (with a link to the formal publication through the relevant DOI); and does not represent the author(s) as endorsing the adaptation of the article or modify the article in such a way as to damage the authors' honor or reputation.
