Biotechnology Letters

, Volume 39, Issue 5, pp 745–750 | Cite as

The structure of a small GTPaseRhoA in complex with PDZRhoGEF and the inhibitor HL47

Original Research Paper



To study the structure of a small GTPaseRhoA in complex with PDZRhoGEF and the inhibitor HL47, and to provide an easier template for R&D of RhoA inhibitor.


Our initial attempts to obtain a binary complex of RhoA with the inhibitor HL47 were unsuccessful probably due to the presence of GDP. By targeting a ternary complex involving the RhoA-specific guanine nucleotide exchange factor PDZRhoGEF, we eliminated GDP and obtained a 2.3 Å structure of the RhoA-PDZRhoGEF-inhibitor HL47 ternary complex.


This structure provides a new template for target-based pharmaceutical design against RhoA.


RhoA-PDZRhoGEF-inhibitor HL47 ternary complex Target-based pharmaceutical design X-ray structure 



The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant Nos. 81202394, 21222211, 21372001 and 91313303), the Ministry of Education of China’s Program for New Century Excellent Talents in University (Grant No. NCET-12-0853), the Young Medical Talents Project of Jiangsu Province, and the Applied Basic Research Programs of Suzhou Sci-tech Bureau, China (Grant No. SYS201219).

Supporting information

Supplementary Table 1—Macromolecule production data for RhoA and PDZRhoGEF.

Supplementary Table 2—Crystallization information.

Supplementary Table 3—Data collection and processing.

Supplementary Table 4—Structure refinement.

Supplementary material

10529_2017_2292_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 17 kb)
10529_2017_2292_MOESM2_ESM.docx (17 kb)
Supplementary material 2 (DOCX 17 kb)
10529_2017_2292_MOESM3_ESM.docx (18 kb)
Supplementary material 3 (DOCX 18 kb)
10529_2017_2292_MOESM4_ESM.docx (18 kb)
Supplementary material 4 (DOCX 17 kb)


  1. Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K (1997) Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275:1308–1311CrossRefPubMedGoogle Scholar
  2. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci USA 98:10037–10041CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) Acta Cryst D66:12–21Google Scholar
  4. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Cryst D50:760–763Google Scholar
  5. D’Arcy A, Bergfors T, Cowan-Jacob SW, Marsh M (2014) Microseed matrix screening for optimization in protein crystallization: what have we learned. Acta Cryst F70:1117–1126Google Scholar
  6. DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, USA.
  7. Deng J, Feng EG, Ma S, Zhang Y, Liu XF, Li HL, Huang H, Zhu J, Zhu WL, Xu S, Miao LY, Liu H, Jiang HL, Li J (2011) Design and synthesis of small molecule RhoA inhibitors: a new promising therapy for cardiovascular diseases. J Med Chem 54:4508–4822CrossRefPubMedGoogle Scholar
  8. Derewenda U, Oleksy A, Stevenson AS, Korczynska J, Dauter Z, Somlyo AP, Otlewski J, Somlyo AV, Derewenda ZS (2004) The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle. Structure 12:1955–1965CrossRefPubMedGoogle Scholar
  9. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Cryst D66:486–501Google Scholar
  10. Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420:629–635CrossRefPubMedGoogle Scholar
  11. Gamblin SJ, Smerdon SJ (1998) GTPase-activating proteins and their complexes. Curr Opin Struct Biol 8:195–201CrossRefPubMedGoogle Scholar
  12. Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9:690–701CrossRefPubMedGoogle Scholar
  13. Ihara K, Muraguchi S, Kato M, Shimizu T, Shirakawa M, Kuroda S, Kaibuchi K, Hakoshima T (1998) Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue. J Biol Chem 273:9656–9666CrossRefPubMedGoogle Scholar
  14. Jaffe AB, Hall A (2005) Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21:247–269CrossRefPubMedGoogle Scholar
  15. Kaibuchi K, Kuroda S, Amano M (1999) Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem 68:459–486CrossRefPubMedGoogle Scholar
  16. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemicai quality of protein structures. J Appl Cryst 26:283–291CrossRefGoogle Scholar
  17. Li DB, Yang GJ, Xu HW, Fu ZX, Wang SW, Hu SJ (2013) Regulation on RhoA in vascular smooth muscle cells under inflammatory stimulation proposes a novel mechanism mediating the multiple-beneficial action of acetylsalicylic acid. Inflammation 36:1403–1414CrossRefPubMedGoogle Scholar
  18. Ma S, Deng J, Li BL, Li XJ, Yan ZW, Zhu J, Chen G, Wang Z, Jiang HL, Miao LY, Li J (2015) Development of second-generation small-molecule RhoA inhibitors with enhanced water solubility, tissue potency, and significant in vivo efficacy. Chem Med Chem 10:193–206CrossRefPubMedGoogle Scholar
  19. Madaule P, Axel R (1985) A novel ras-related gene family. Cell 41:31–40CrossRefPubMedGoogle Scholar
  20. Matthews BW (1968) Solvent content of protein crystals. J Mol Biol 33:491–497CrossRefPubMedGoogle Scholar
  21. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674CrossRefPubMedPubMedCentralGoogle Scholar
  22. Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA (2011) Acta Cryst D67:355–367Google Scholar
  23. Narumiya S (1996) The small GTPase Rho: cellular functions and signal transduction. J Biochem 120:215–228CrossRefPubMedGoogle Scholar
  24. Takai Y, Sasaki T, Tanaka K, Nakanishi H (1995) Rho as a regulator of the cytoskeleton. Trends Biochem Sci 20:227–231CrossRefPubMedGoogle Scholar
  25. Vagin A, Teplyakov A (2010) Molecular replacement with MOLREP. Acta Cryst D66:22–25Google Scholar
  26. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Acta Cryst D67:235–242Google Scholar
  27. Yin Y, Lin L, Ruiz C, Khan S, Cameron MD, Grant W, Pocas J, Eid N, Park H, Schroter T (2013) Synthesis and biological evaluation of urea derivatives as highly potent and selective rho kinase inhibitors. J Med Chem 56:3568–3581CrossRefPubMedGoogle Scholar
  28. Zhang Y, Deng J, Ma S, Xue L, Zhu J, Zhu WL, Jiang HL, Li J, Miao LY (2012) The effect of first-in-class small molecule RhoA inhibitor, HL07, on the phenylephrine-induced artery contraction. Curr Pharm Des 27:4258–4264CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Department of Clinical PharmacologyThe First Affiliated Hospital of Soochow UniversitySuzhouPeople’s Republic of China
  2. 2.Wuxi Biortus Biosciences Co.LtdJiangyinPeople’s Republic of China
  3. 3.Shanghai Key Laboratory of New Drug DesignSchool of Pharmacy East China University of Science and TechnologyShanghaiPeople’s Republic of China

Personalised recommendations