Structural biology of bacterial response regulator proteins and their complexes with cognate ligands

Two bacterial response regulator systems were studied in this thesis. NMR
spectroscopy and X-ray crystallography were used to determine the structures of both
domains of the antibiotic sensor TipAL, as well as of the c-di-GMP receptor PA4608,
in complex with cognate ligands. By comparison with structures of the free proteins,
we found that ligand binding induced biologically relevant structural rearrangements
in all proteins studied.
The putative multidrug resistance gene tipA of the soil bacterium Streptomyces
lividans codes for the transcription factor TipAL, which comprises two domains: a
DNA-binding domain named TipAN, and a ligand-binding domain named TipAS,
which is capable of recognizing and binding a diverse group of macrocyclic
thiopeptide antibiotics. After antibiotic binding, TipAL binds to promoter DNA and
activates transcription. In order to elucidate the specificity and flexibility of antibiotic
recognition as well as the mechanism of transcriptional activation, the two domains of
TipAL were studied separately.
We have determined the structures of TipAS with bound promothiocin A or
nosiheptide antibiotics by NMR spectroscopy. The N-terminal part of the TipAS
sequence, which is flexible and unstructured in free TipAS, forms three new helices in
both complexes, burying the bound antibiotics. Considering that the newly formed
helices form the connection between the TipAS and TipAN domains, we propose that
the formation of additional secondary structure forms the basis of transcriptional
activation by TipAL after ligand binding. The TipAS complexes with promothiocin A
and nosiheptide are similar, but differ in the dynamics of the newly formed helices;
the smaller ligand, promothiocin A, appears to leave more room for movement of
TipAS.
The structure of TipAN in complex with a fragment of tipA promoter DNA was
solved by X-ray crystallography. TipAN binds to the symmetric promoter as a dimer,
which is held together by a long, antiparallel coiled coil. In contrast to homologous
proteins, TipAN does not bend and twist bound DNA, which is a prerequisite for
transcriptional activation by other proteins of the same family. This indicates that the
activated TipAS domain is required for transcriptional activation by TipAL.
C-di-GMP is a second messenger molecule that appears to be ubiquitous in, and
unique to, the bacterial kingdom. It generally controls the switch from motile, singlecell
lifestyles to surface-attached, multicellular communities such as biofilms. The
natural receptors of c-di-GMP are the PilZ domain proteins, which include PA4608 in
Pseudomonas aeruginosa. We have solved the NMR solution structure of PA4608 in
complex with c-di-GMP. C-di-GMP binds to the protein as an intercalated dimer, displacing the C-terminal 310 helix found in the apo form. The N-terminal part of
PA4608, which contains the highly conserved RxxxR motif and which is flexible and
unstructured before ligand binding, contacts the distal side of c-di-GMP.