Miljan Simonovic, Ph.D.
Jane Coffin Postdoctoral Fellow

Center for Structural Biology
Department of Molecular Biophysics & Biochemistry
Yale University
266 Whitney Avenue, 415 Bass Center
New Haven, CT 06520-8114

Phone:  (203) 432-5627
Fax:  (203) 432-3282

Email: miljan.simonovic {at} yale.edu

Structural study of cotranslational translocation

Summary:

Ribosome-catalyzed translation of mRNA into protein is a fundamental process in living cells. An essential part of this event is cotranslational translocation, a process by which many secretory and membrane proteins are synthesized and simultaneously transported across the lipid bilayer. While the sequence of these events is well described by biochemical studies (Fig. 1), the molecular mechanism underlying this process is not understood.

In eukaryotes, cotranslational translocation takes place on the ribosomes attached to the translocon protein complex (Fig. 2) located in the endoplasmic reticulum membrane. In prokaryotes, the same process occurs on the inner cell membrane and is employed mostly for membrane protein synthesis. Only the initial step of cotranslational translocation is common to both membrane and secretory proteins. During this step the nascent polypeptide chain inserts into the translocon pore resulting in tight ribosome-translocon interactions, which are mediated by large subunit rRNA. High-resolution structural analysis of the initial step is crucial for understanding the general mechanism of cotranslational translocation. Here I am studying the initial step of cotranslational translocation of the archebacterial ribosome-translocon complex using X-ray crystallographic techniques. Because ribosome-translocon interactions are evolutionarily conserved, results of this study will provide an understanding of eukaryotic cotranslational translocation as well.

 

 

Fig. 1 – The mechanism of cotranslational translocation in bacteria (Driessen, A.J.M et al., NSB, 8: 492-498, 2001).

 

Fig. 2 – The crystal structure of an archaeal translocon (van den Berg, B., Clemons, W. M. Jr. et al., Nature, 2003).

 

Fig. 3 – The Cryo-EM structure of the ribosome-translocon complex (Beckmann, R. et al., Science, 278: 2123-2126, 1997).

Previous research:

I obtained doctoral degree at the University of Illinois at Chicago - Department of Biochemistry and Molecular Biology. There I studied the inhibitory mechanism of serpins, the mechanism of bacterial signal transduction and ligand binding by LRP receptor using X-ray crystallography techniques:

  • Crystal structure of the viral serpin crmA in cleaved form (PDB ID: 1C80 and 1M93) was the first structure of a serpin that inhibits both cysteine and serine proteinases. The structure suggested that crmA employs the same inhibitory mechanism as other inhibitory serpins and also revealed a novel structural feature designated as strand 1’A (s1’A). A non-isomorphous structure of the RCL-cleaved crmA and NMR-HSQC experiments on a series of crmA mutants revealed high flexibility of the strand 1’A.
  • Crystal structure of the human non-inhibitory serpin PEDF from retina (PDB ID: 1IMV) was the first of a biologically active non-inhibitory serpin and it revealed the three-dimensional organization of biologically important sites as well as striking asymmetric charge distribution unprecedented in the serpin superfamily. PEDF is potent neurothrophic and anti-angiogenic factor in the human central nervous system and retina, which mechanism of action is not understood in its entirety. The structure suggested new experiments aimed at deciphering the mechanism of PEDF action.
  • X-ray structures of the Pittsburgh variant of a1-proteinase inhibitor (a1PI-P) and that of the non-covalent complex between a1PI-P and inactive trypsin (PDB ID: 1OO8 and 1OPH). a1PI-P is a poor inhibitor of human neutrophil elastase due to M to R point mutation at the P1 position within the reactive center loop (RCL). However, a1PI-P is a very good thrombin inhibitor and as such is able to disrupt blood coagulation homeostasis. The crystal structure of a1PI-P and that of the non-covalent complex between a1PI-P and inactive trypsin (S195A) showed that canonical-like interactions between the serpin RCL and the proteinase active site determine serpin specificity.
  • High resolution structure of the bacterial response regulator CheY (PDB ID: 1JBE) has shown that apo-CheY exists as a mixture of inactive and meta-active conformers. Very high resolution allowed us to study an unusual posttranslational modification, the succinimidyl ring, formed between the side-chain of Asp75 and the backbone amide of Gly76.
  • Crystal structure of the human complement-like repeat 7 (CR7) domain from LRP receptor (PDB ID: 1J8E) provided more insights into the mechanism of LRP function and showed that complement-like repeats, despite their similar fold, have different binding surfaces and charge distribution.

Publications:

  1. Simonovic M, Zhang Z, Cianci C, Steitz TA, Morrow JD (2006) Structure of the calmodulin aII-spectrin complex provides insight into the regulation of cell plasticity. (in press).
  2. Simonovic M, Volz K, Salvessen G, Gettins PG (2005) Lack of involvement of strand s1'A of the viral serpin CrmA in anti-apoptotic or caspase-inhibitory functions. Arch. Biophys. Biochem, 440, 1-9.
  3. Viswanathan VK, Koutsouris A, Lukic S, Pilkinton M, Simonovic I, Simonovic M, Hecht G (2004) Comparative analysis of EspF from enteropathogenic and enterohemorrhagic Escherichia coli in alteration of epithelial barrier function. Infection and Immunity, 72, 3218-3227.
  4. Dementiev A, Simonovic M, Volz K, Gettins PG. (2003) Canonical inhibitor-like interactions explain reactivity of alpha 1-PI Pittsburgh and antithrombin with proteinases. J. Biol. Chem., 278, 37881-37887.
  5. Gettins PG, Simonovic M, Volz K (2002) Pigment epithelium-derived factor (PEDF), a serpin with potent anti-angiogenic and neurite outgrowth-promoting properties. Biol. Chem., 383, 1677-1682.
  6. Simonovic M and Volz K (2002) Atomic resolution structure of a succinimide intermediate in E. coli CheY. J. Mol. Biol., 322, 663-667.
  7. Simonovic M, Gettins PG, Volz K (2001) Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor. PNAS, 98, 11131-11135.
  8. Simonovic M and Volz K (2001) A distinct meta-active conformation in the 1.1 Å resolution structure of wild-type, apo-CheY. J. Biol. Chem., 276, 28637-28640.
  9. Simonovic M, Dolmer K, Huang W, Strickland DK, Volz K, Gettins PG (2001) X-ray structure and calcium affinity of ligand-binding repeat CR7 from LRP. Comparison with related domains from LRP and LDL receptor. Biochemistry, 40, 15127-15134.
  10. Simonovic M, Gettins PG, Volz K (2000) Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition. Protein Sci., 9, 1423-1427.
  11. Simonovic M, Gettins PG, Volz K (2000) Crystallization and preliminary X-ray diffraction analysis of a recombinant cysteine-free mutant of CrmA. Acta Cryst. D, 56, 1440-1442.
  12. Ma J, Simonovic M, Qian R, Colley KJ (1999) Sialyltransferase isoforms are phosphorylated in the cis-medial Golgi on serine and threonine residues in their luminal sequences. J. Biol. Chem., 274, 8046-8052.
  13. Simonovic M, Soskic V, Joksimovic J (1999) Purification of Gai,2 subunit from bovine brain by affinity chromatography. Jug. Med. Bioh., 17, 385-390.
  14. Simonovic M, Soskic V, Joksimovic J (1998) Quantification of human dopamine D2S receptor interactions with Gai,1,2- and Gao-proteins. Neurochemistry Int., 33, 271-275.