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

, Yun-Tzai Lee

1,2

, Shih-Yun Chen

, Yei-Chen Lai

, Meng-Ru Ho

, Yun-Wei

Chiang

Shang-Te Danny Hsu

1,2

Academia Sinica, Taipei, Taiwan,

National Taiwan University, Taipei, Taiwan,

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.