Validation of Nanobodies as ‘‘conformational locks’’ to stabilize the low affinity MnmE-GidA protein complex

Samenvatting

Transient PPIs are intervenient in major cellular processes and are considered
to be interesting drug targets. Nevertheless, little is known about transient PPIs
because they are difficult to study due to their rapid association-dissociation rate.
Here, we attempted to generate Nanobodies able to stabilize the transient interaction
between MnmE/GidA complex - an E. coli protein complex involved in the
modification of the wobble uridine from tRNAs reading codons from split codon
boxes. The structure of MnmE and GidA individually is known. However, several
attempts to crystallize the complex failed.
Nanobodies have shown to be great crystallization aids for stabilization of
intrinsically flexible proteins or membrane proteins. Due to the sensitive requirements
for protein crystallization, Nanobodies could serve as crystallization chaperones
prompting the crystallization of MnmE/GidA complex by stabilizing this transient
interaction.
To this, we immunized two llamas and generated two separated Nanobody
phage display libraries. We were able to capture MnmE/GidA complex and perform
alternate monomer immobilization panning that allowed the selection of Nanobodies
recognizing epitopes exclusive from the complex. By ELISA screening and analytical
gel filtration, we were able to prove that Nanobody CA4997 is able to bind to the
complex specific epitopes and to GidA. Yet, a comparison of the MnmE/GidA
complex in presence and absence of Nanobody is still necessary to prove that the KD
of MnmE/GidA can decrease in presence of this or another specific Nanobody. A
stabilized interaction could facilitate the crystallization of MnmE/GidA in the
complex form and ultimately retrieve the structure of this complex at atomic
resolution by protein crystallography. In the future, this strategy can be used to
stabilize other transient interactions prompting their study and lead to innovative
discoveries.

Datum prijs	1 sep. 2011
Originele taal	English
Begeleider	Wim Versées (Promotor)