Skip to main content
SpringerLink
Log in

Detection of structural and conformational changes in ALS-causing mutant profilin-1 with hydrogen/deuterium exchange mass spectrometry and bioinformatics techniques

  • Original Article
  • Published:
Metabolic Brain Disease Aims and scope Submit manuscript

Abstract

The hydrogen/deuterium exchange (HDX) is a reliable method to survey the dynamic behavior of proteins and epitope mapping. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a quantifying tool to assay for HDX in the protein of interest. We combined HDX-MALDI-TOF MS and molecular docking/MD simulation to identify accessible amino acids and analyze their contribution into the structural changes of profilin-1 (PFN-1). The molecular docking/MD simulations are computational tools for enabling the analysis of the type of amino acids that may be involved via HDX identified under the lowest binding energy condition. Glycine to valine amino acid (G117V) substitution mutation is linked to amyotrophic lateral sclerosis (ALS). This mutation is found to be in the actin-binding site of PFN-1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN-1 protein dynamic behavior using purified wild type and mutant PFN-1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN-1WT and PFN-1G117V at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN-1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN-1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFNG117V. This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN-1s’ dimerization, oligomerization, and aggregation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files as a movie can be found in the link shown here: https://doi.org/10.6084/m9.figshare.13551218

https://doi.org/10.6084/m9.figshare.14447229.v1

https://doi.org/10.6084/m9.figshare.14447292.v1

Abbreviations

ALS:

amyotrophic lateral sclerosis

PFN-1:

Profilin-1

HDX:

hydrogen/deuterium exchange

MALDI-TOF MS:

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

MD:

Molecular Dynamics

PDB:

Protein Data Bank

References

  • Alkam D, Feldman EZ, Singh A, Kiaei M (2017) Profilin1 biology and its mutation, actin (g) in disease. Cell Mol Life Sci 74(6):967–981

    CAS  PubMed  Google Scholar 

  • Brown RH, Al-Chalabi A (2017) Amyotrophic lateral sclerosis. N Engl J Med 377(2):162–72

  • Busenlehner LS, Armstrong RN (2005) Insights into enzyme structure and dynamics elucidated by amide H/D exchange mass spectrometry. Arch Biochem Biophys 433(1):34–46

    CAS  PubMed  Google Scholar 

  • Chen Y, Huang R, Chen K, Song W, Zhao B, Chen X, Yang Y, Yuan L, Shang H-F (2013) PFN-1 mutations are rare in Han Chinese populations with amyotrophic lateral sclerosis. Neurobiol Aging 34(7):1922. e1921-1922. e1925

  • Englander SW (2006) Hydrogen exchange and mass spectrometry: A historical perspective. J Am Soc Mass Spectrom 17(11):1481–1489

    CAS  PubMed  PubMed Central  Google Scholar 

  • Englander JJ, Del Mar C, Li W, Englander SW, Kim JS, Stranz DD, Hamuro Y, Woods VL (2003) Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry. Proc Natl Acad Sci 100(12):7057–7062

  • Fedorov A, Pollard T, Almo SC (1994) Purification, characterization and crystallization of human platelet profilin expressed in Escherichia coli. J Mol Biol 241(3):480–482

    CAS  PubMed  Google Scholar 

  • Ferrin TE, Huang CC, Jarvis LE, Langridge R (1988) The MIDAS display system. J Mol Graph 6(1):13–27

    CAS  Google Scholar 

  • Figueroa ID, Russell DH (1999) Matrix-assisted laser desorption ionization hydrogen/deuterium exchange studies to probe peptide conformational changes. J Am Soc Mass Spectrom 10(8):719–731

    CAS  PubMed  Google Scholar 

  • Fil D, DeLoach A, Yadav S, Alkam D, MacNicol M, Singh A, Compadre CM, Goellner JJ, O’Brien CA, Fahmi T (2017) Mutant Profilin1 transgenic mice recapitulate cardinal features of motor neuron disease. Hum Mol Genet 26(4):ddw429

    Google Scholar 

  • Frisch M, Trucks G, Schlegel H, Scuseria G, Robb M, Cheeseman J, Montgomery J, Vreven T (2004) Udin KN, Burant JC, et al. Gaussian 03, Version C. 02. Gaussian, Inc, Wallingford

  • Gaussian03 R.B (2004) 05, Frisch MJ et al. Gaussian Inc, Wallingford

  • Hardiman O, Al-Chalabi A, Chio A, Corr EM, Logroscino G, Robberecht W, Shaw PJ, Simmons Z, Van Den Berg LH (2017) Amyotrophic lateral sclerosis. Nat Rev Dis Primers 5(1):1–9

    Google Scholar 

  • Hentze N, Mayer MP (2013) Analyzing protein dynamics using hydrogen exchange mass spectrometry. JoVE (81):e50839

  • Hospital A, Goñi JR, Orozco M, Gelpí JL (2015) Molecular dynamics simulations: advances and applications. Adv Appl Bioinforma Chem 8:37

    Google Scholar 

  • Kiaei M, Balasubramaniam M, Kumar VG, Reis RJS, Moradi M, Varughese KI (2018) ALS-causing mutations in profilin-1 alter its conformational dynamics: a computational approach to explain propensity for aggregation. Sci Rep 8(1):1–10

    CAS  Google Scholar 

  • Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, Li Q, Shoemaker BA, Thiessen PA, Yu B (2019) PubChem 2019 update: improved access to chemical data. Nucleic Acids Res 47(D1):D1102–D1109

    PubMed  Google Scholar 

  • Kochert BA, Iacob RE, Wales TE, Makriyannis A, Engen JR (2018) Hydrogen-deuterium exchange mass spectrometry to study protein complexes. Protein Complex Assembly, Springer, Berlin, pp 153–171

  • Kovalchuk MO, Heuberger JA, Sleutjes BT, Ziagkos D, van den Berg LH, Ferguson TA, Franssen H, Groeneveld GJ (2018) Acute effects of riluzole and retigabine on axonal excitability in patients with amyotrophic lateral sclerosis: a randomized, double-blind, placebo‐controlled, crossover trial. Clin Pharmacol Ther 104(6):1136–1145

    CAS  PubMed  Google Scholar 

  • Krishnan K, Holub O, Gratton E, Clayton AH, Cody S, Moens PD (2009) Profilin interaction with phosphatidylinositol (4, 5)-bisphosphate destabilizes the membrane of giant unilamellar vesicles. Biophys J 96(12):5112–5121

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lambrechts A, Jonckheere V, Dewitte D, Vandekerckhove J, Ampe C (2002) Mutational analysis of human profilin I reveals a second PI (4, 5)-P 2 binding site neighbouring the poly (L-proline) binding site. BMC Biochem 3(1):12

    PubMed  PubMed Central  Google Scholar 

  • Lee S, Kim H-J (2015) Prion-like mechanism in amyotrophic lateral sclerosis: are protein aggregates the key? Exp Neurobiol 24(1):1–7

    PubMed  Google Scholar 

  • Lemkul J (2018) From proteins to perturbed Hamiltonians: A suite of tutorials for the GROMACS-2018 molecular simulation package [article v1. 0]. Living J Comput Mol Sci 1(1):5068

  • Leurs U, Mistarz UH, Rand KD (2015) Getting to the core of protein pharmaceuticals–Comprehensive structure analysis by mass spectrometry. Eur J Pharm Biopharm 93:95–109

    CAS  PubMed  Google Scholar 

  • Lodowski DT, Palczewski K, Miyagi M (2010) Conformational changes in the G protein-coupled receptor rhodopsin revealed by histidine hydrogen – Deuterium exchange. Biochemistry 49(44):9425–9427

    CAS  PubMed  Google Scholar 

  • Ludolph A, Drory V, Hardiman O, Nakano I, Ravits J, Robberecht W, Shefner J (2015) A revision of the El Escorial criteria-2015. Amyotroph Lateral Scler Frontotemporal Degener 16(5–6):291–2

  • Maier CS, Deinzer ML (2005) Protein conformations, interactions, and H/D exchange. Methods Enzymol 402:312–360

    CAS  PubMed  Google Scholar 

  • Majumdar R, Middaugh CR, Weis DD, Volkin DB (2015) Hydrogen-deuterium exchange mass spectrometry as an emerging analytical tool for stabilization and formulation development of therapeutic monoclonal antibodies. J Pharm Sci 104(2):327–345

    CAS  PubMed  Google Scholar 

  • Metzler WJ, Farmer BT, Constantine KL, Friedrichs MS, Mueller L, Lavoie T (1995) Refined solution structure of human profilin I. Protein Sci 4(3):450–459

    CAS  PubMed  PubMed Central  Google Scholar 

  • Millul A, Beghi E, Logroscino G, Micheli A, Vitelli E, Zardi A (2005) Survival of patients with amyotrophic lateral sclerosis in a population-based registry. Neuroepidemiology 25(3):114–119

    CAS  PubMed  Google Scholar 

  • Montalvao RW, Cavalli A, Salvatella X, Blundell TL, Vendruscolo M (2008) Structure determination of protein – protein complexes using NMR chemical shifts: case of an endonuclease colicin – immunity protein complex. J Am Chem Soc 130(47):15990–15996

    CAS  PubMed  Google Scholar 

  • Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nekouei M, Ghezellou P, Aliahmadi A, Arjmand S, Kiaei M, Ghassempour A (2018) Changes in biophysical characteristics of PFN-1 due to mutation causing amyotrophic lateral sclerosis. Metabolic Brain Dis 33(6):1975–1984

    CAS  Google Scholar 

  • Ngan CH, Bohnuud T, Mottarella SE, Beglov D, Villar EA, Hall DR, Kozakov D, Vajda S (2012) FTMAP: extended protein mapping with user-selected probe molecules. Nucleic Acids Res 40(W1):W271–W275

    CAS  PubMed  PubMed Central  Google Scholar 

  • Palashoff MH (2008) Determining the specificity of pepsin for proteolytic digestion. Northeastern University, Boston

  • Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612

    CAS  PubMed  Google Scholar 

  • Pirrone GF, Wang H, Canfield N, Chin AS, Rhodes TA, Makarov AA (2017) Use of MALDI-MS combined with differential hydrogen–deuterium exchange for semiautomated protein global conformational screening. Anal Chem 89(16):8351–7

  • Robertson MJ, Tirado-Rives J, Jorgensen WL (2015) Improved peptide and protein torsional energetics with the OPLS-AA force field. J Chem Theory Comput 11(7):3499–3509

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rozbesky D, Man P, Kavan D, Chmelik J, Cerny J, Bezouska K, Novak P (2012) Chemical cross-linking and H/D exchange for fast refinement of protein crystal structure. Anal Chem 84(2):867–870

    CAS  PubMed  Google Scholar 

  • Sadr AS, Eslahchi C, Ghassempour A, Kiaei M (2021) In silico studies reveal structural deviations of mutant profilin-1 and interaction with riluzole and edaravone in amyotrophic lateral sclerosis. Sci Rep 25(1):1–4

    Google Scholar 

  • Schutt CE, Myslik JC, Rozycki MD, Goonesekere NC, Lindberg U (1993) The structure of crystalline profilin–β-actin. Nature 365(6449):810–816

    CAS  PubMed  Google Scholar 

  • Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860

    CAS  PubMed  Google Scholar 

  • Thomsen R, Christensen MH, MolDock: a new technique for high-accuracy molecular docking, J Med Chem 49 (2006) 3315–3321, https://doi.org/10.1021/jm051197e

    Article  CAS  PubMed  Google Scholar 

  • Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455–61

  • Villanueva J, Canals F, Villegas V, Querol E, Avilés FX (2000) Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins. FEBS Lett 472(1):27–33

    CAS  PubMed  Google Scholar 

  • Wales TE, Engen JR (2006) Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom Rev 25(1):158–170

    CAS  PubMed  Google Scholar 

  • Wei H, Mo J, Tao L, Russell RJ, Tymiak AA, Chen G, Iacob RE, Engen JR (2014) Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discov Today 19(1):95–102

    CAS  PubMed  Google Scholar 

  • Weis DD (2016) Hydrogen exchange mass spectrometry of proteins: fundamentals, methods, and applications. Wiley, Hoboken

  • West GM, Chien EY, Katritch V, Gatchalian J, Chalmers MJ, Stevens RC, Griffin PR (2011) Ligand-dependent perturbation of the conformational ensemble for the GPCR β2 adrenergic receptor revealed by HDX. Structure 19(10):1424–1432

    CAS  PubMed  PubMed Central  Google Scholar 

  • Witke W (2004) The role of profilin complexes in cell motility and other cellular processes. Trends Cell Biol 14(8):461–469

    CAS  PubMed  Google Scholar 

  • Wu C-H, Fallini C, Ticozzi N, Keagle PJ, Sapp PC, Piotrowska K, Lowe P, Koppers M, McKenna-Yasek D, Baron DM (2012) Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature 488(7412):499–503

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xiao K, Chung J, Wall A (2015) The power of mass spectrometry in structural characterization of GPCR signaling. J Recept Signal Transduction 35(3):213–219

    CAS  Google Scholar 

  • Xiao K, Zhao Y, Choi M, Liu H, Blanc A, Qian J, Cahill TJ, Li X, Xiao Y, Clark LJ (2018) Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping. Nat Protoc 13(6):1403–1428

    CAS  PubMed  Google Scholar 

  • Zhang Z, Smith DL (1993) Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci 2(4):522–531

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang L, Bell DR, Luan B, Zhou R (2019) Exploring the binding mechanism between human profilin (PFN-1) and polyproline-10 through binding mode screening. J Chem Phys 150(1):015102

    PubMed  Google Scholar 

  • Zhou M, Robinson CV (2014) Flexible membrane proteins: functional dynamics captured by mass spectrometry. Curr Opin Struct Biol 28:122–130

    CAS  PubMed  Google Scholar 

Download references

Funding

We acknowledge the support from RockGen Therapeutics, LLC. There is no other financial support to disclose.

Author information

Author notes

  1. Ahmad Shahir Sadr and Zahra Abdollahpour contributed equally to this work.

Authors and Affiliations

  1. Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran

    Ahmad Shahir Sadr, Zahra Abdollahpour, Atousa Aliahmadi, Mina Nekouei & Alireza Ghassempour

  2. Computer Science Department, Mathematical Sciences Faculty, Shahid Beheshti University, Tehran, Iran

    Ahmad Shahir Sadr & Changiz Eslahchi

  3. School of Biological Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran

    Changiz Eslahchi

  4. Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA

    Mahmoud Kiaei

  5. Department of Neurology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA

    Mahmoud Kiaei

  6. Department of Geriatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA

    Mahmoud Kiaei

  7. RockGen Therapeutics, LLC, Little Rock, AR, 72205, USA

    Lily Kiaei & Mahmoud Kiaei

Authors
  1. Ahmad Shahir Sadr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zahra Abdollahpour
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Atousa Aliahmadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Changiz Eslahchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mina Nekouei
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lily Kiaei
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mahmoud Kiaei
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alireza Ghassempour
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The concept and idea of the study was designed by MK, CE, ZA, MN, ASS, AG. The experiments were designed and carried out by ZA, AA, MN, ASS. The data were analyzed and evaluated by MK, CE, ASS, AG. The manuscript was written by MK, CE, ASS, ZA, LK, AG.

Corresponding authors

Correspondence to Mahmoud Kiaei or Alireza Ghassempour.

Ethics declarations

Disclosure of potential conflicts of interest

Dr. Kiaei is the founder, President and CEO of RockGen Therapeutics, LLC., and have a financial interest in the technology discussed in this publication. These financial interests have been reviewed and approved in accordance with the RockGen conflict of interest policies. Other authors have no conflict.

Research involving human participants and/or animals

There were no human participants or animals used in this study.

Informed consent

N/A.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Mahmoud Kiaei and Alireza Ghassempour are co-senior authors.

Supplementary Information

ESM 1

Table 1Sa. Distinct conformational clusters finding, out of 220 runs of acetaldehyde over PFN-1G117V, using RMSD -tolerance of 2.0 Å with Autodock 4. Table 1Sb. Docking Results Obtained by Autodock Vina. Table 1Sc. MolDock score; Re-rank score, the hydrogen bond energy of the docked compound, docking Score and Similarity Score by Molegro virtual docker. Table 2Sa. Distinct conformational clusters finding, out of 220 runs of acetaldehyde over PFN-1WT, using RMSD -tolerance of 2.0 Å. Table 2Sb. Docking Results Obtained by Autodock Vina. Table 2Sc. MolDock score; Re-rank score, the hydrogen bond energy of the docked compound, docking Score and Similarity Score by Molegro virtual docker. Table 3Sa. Distinct conformational clusters finding, out of 220 runs of benzaldehyde over PFN-1G117V, using RMSD -tolerance of 2.0 Å. Table 3Sb. Docking Results Obtained by Autodock Vina. Table 3Sc. MolDock score; Re-rank score, the hydrogen bond energy of the docked compound, by Molegro virtual docker. Table 4Sa. Distinct conformational clusters founding, out of 220 runs of benzaldehyde over PFN-1WT, using RMSD-tolerance of 2.0 Å. Table 4Sb. Docking Results Obtained by Autodock Vina. Table 4Sc. MolDock score; Re-rank score, the hydrogen bond energy of the docked compound, by Molegro virtual docker. Figure 1S. Coomassie blue SDS-PAGE related to the IPTG concentration optimization of PFN-1WT and PFN-1G117V. (a) Optimization of IPTG concentration related to PFN-1WT in different IPTG molarity. From left to right: Ladder, before induction (BI) ,0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 m M and 1 mM of IPTG during 20 h after induction. (b) Optimization of IPTG concentration related to PFN-1G117V in different IPTG molarity, from left to right: before induction, Ladder, 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1 mM of IPTG during 20 h after induction. Black arrow points to protein band, PFN-1, expressed the most at 0.4 mM IPTG in the case of WT PFN-1 and at 0.8 mM IPTG for PFN-1G117V. Figure 2S. Coomassie blue stained SDS-PAGE related to the time of induction optimization of PFN-1WT and PFN-1G117V, from left to right: Ladder, before induction (BI), 2 h, 4 and 20 h after induction (PFN-1WT), before induction (BI), 2 h, 4 and 20 h after induction (PFN-1G117V). Black arrow points to protein band, PFN-1G117V, that required 20 h of induction for either WT or mutant PFN-1. Figure 3S. Sonication of the bacterial cells at 20 °C related to PFN-1WT and PFN-1G117V. From left to right; (a) Ladder, before induction (BI), before sonic (BS), after sonic (AS), pellet after centrifugation in 2000 rpm (P2000), supernatant after centrifugation in 2000 rpm (S2000), supernatant after centrifugation in 13,000 rpm (S13000) and pellet after centrifugation in 13,000 rpm (P13000). (b) Ladder, before induction (BI), before sonic (BS), after sonic (AS), pellet after centrifugation in 2000 rpm (P2000), supernatant after centrifugation in 2000 rpm (S2000), supernatant after centrifugation in 13,000 rpm (S13000) and pellet after centrifugation in 13,000 rpm (P13000). Arrow points to most pure PFN-1 at S13000 rpm. (DOCX 2.72 MB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sadr, A.S., Abdollahpour, Z., Aliahmadi, A. et al. Detection of structural and conformational changes in ALS-causing mutant profilin-1 with hydrogen/deuterium exchange mass spectrometry and bioinformatics techniques. Metab Brain Dis 37, 229–241 (2022). https://doi.org/10.1007/s11011-021-00763-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11011-021-00763-y

Keywords

Search

Navigation