145
Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery Poster Session II
92-POS
Board 45
Going through the Motions: Network Analyses of Molecular Dynamics Simulations Reveal
Conserved Intramolecular Communication Pathways within EF-Tu Responsible for
Ribosome Dependent GTPase Activation and Nucleotide Exchange.
Evan Mercier, Dylan Girodat, Fan Mo,
Hans-Joachim Wieden
.
University of Lethbridge, Lethbridge, Canada.
During the elongation cycle of bacterial protein synthesis elongation factor (EF) Tu delivers
aminoacyl(aa)-tRNAs to the ribosome in a GTP-dependent manner. EF-Tu functions as a
checkpoint, correct codon recognition is required to send an activating signal from the decoding
centre on the 30S ribosomal subunit to the G domain of EF-Tu bound to the 50S ribosomal
subunit. Following dissociation, EF-Tu•GDP is recycled into its GTP-bound form through the
interaction with its nucleotide exchange factor EF-Ts. Both functions require the transmission of
signals to facilitate either GTP hydrolysis or nucleotide release. Little is known about the
dynamic features governing signal transmission within a highly conserved protein such as EF-Tu
and its particular evolutionary constraints.
Here, molecular dynamics simulations of EF-Tu and variants with single amino acid
substitutions were performed and subsequently interrogated using network analysis methods.
Using these networks we identify communication pathways essential for efficient GTPase
activation on the ribosome as well as EF-Ts-catalyzed nucleotide exchange. We find that single
amino acid substitutions in EF-Tu can change the network organization dramatically and result
in significantly reduced communication between domains across a set of universally conserved
interdomain bridges. To validate our findings we have performed Michaelis-Menten analyses of
ribosome-stimulated GTP hydrolysis in EF-Tu. Consistent with our network analysis, these
results reveal that amino acid substitutions in domain II reduce the stimulatory effect of the 70S
ribosome five-fold, while not affecting 50S-dependent stimulation. Variants of EF-Tu that
disrupt the interdomain bridges reduce the stimulatory effect of the 70S ribosome to a similar
extent. Analyses of the EF-Tu•EF-Ts complex revealed a different set of communication
pathways promoting nucleotide exchange in EF-Tu which were validated using rapid-kinetics
techniques.
