Conformational Ensembles from Experimental Data and Computer Simulations

Conformational Ensembles from Experimental Data and Computer Simulations

Sunday Speaker Abstracts

Structural Basis of Substrate Recognition and Chaperone Activity of Ribosome-associated Trigger Factor Regulated by Monomer-dimer Equilibrium Chih-Ting Huang 1 , Yun-Tzai Lee 1,2 , Shih-Yun Chen 1 , Yei-Chen Lai 3 , Meng-Ru Ho 1 , Yun-Wei Chiang 3 , Shang-Te Danny Hsu 1,2 . 1 Academia Sinica, Taipei, Taiwan, 2 National Taiwan University, Taipei, Taiwan, 3 National Tsing Hua University, Hsinchu, Taiwan. Trigger factor (TF) is a highly conserved bacterial chaperone that binds as a monomer via the ribosome binding domain (RBD) to the exit tunnel of the ribosome to facilitate co-translational folding of nascent polypeptide chains. Free TF however, exists in a monomer-dimer equilibrium in solution with a dissociation constant comparable to its physiological concentration. Using fluorescence anisotropy and nuclear magnetic resonance (NMR) spectroscopy, we established quantitatively that TF preferentially recognizes peptide segments enriched with aromatic and positively charged amino acids to form fuzzy complexes through binding to four distinct sites in TF. Paramagnetic NMR analysis indicated that three of these substrate binding sites within TF are sequestered upon dimer formation mediated by RBD. Small angle X-ray scattering (SAXS) deomnstrated that the dimeric assembly of TF in solution deviates significantly from the previously reported crystal structure. We therefore devised an integrated approach using structural restrains derived from paramagnetic NMR, pulsed electron paramagnetic resonance, chemical cross-linking and SAXS to determine the solution structure of TF dimer in an antiparallel configuration. Our structural and functional analyses suggested that the dynamic equilibrium of the oligomeric state of TF is important for maintaining the balance between substrate binding and chaperone activities on the one hand, and preventing excessive exposure of hydrophobic surface on the other hand. Furthermore, the RBD of TF plays a dual role in regulating the three-state equilibrium between self-association and ribosome binding.

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