[Todos] Seminarios DQIAQF – INQUIMAE, lunes 18 de julio - 13 hs.

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Seminarios DQIAQF – INQUIMAE, lunes 18 de julio - 13 hs.

Aula de Seminarios INQUIMAE - DQIAQF
Facultad de Ciencias Exactas y Naturales
Ciudad Universitaria - Pab. 2  -  Piso 3

*"Histidine kinases in bacterial signaling: proteins on the move"*

Dr. Alejandro Buschiazzo

Research Scientist

Unit of Protein Crystallography

Institut Pasteur de Montevideo

* *
Abstract :
DesK is a membrane-bound histidine kinase (HK) from Bacillus subtilis, able
to sense the order of membrane lipids when cells are subjected to cold
shock, ultimately behaving as a molecular thermometer. Although the
relevance of sensor HKs in signal transduction is well established, we still
do not understand at the molecular level how HKs transduce input signal
information to regulate their output catalytic activities. We address this
issue by using a combination of structural and biochemical approaches. We
have determined eight crystal structures of the intracytoplasmic catalytic
core of DesK, including the wild-type, the phosphorylated form, and point
mutants that retain particular functional traits.
Structural analyses show that DesK has been trapped in three conformational
states that correspond to alternate functions of the protein along the
signaling pathway [1]. By comparing the 3D structures of a single HK in
different functional configurations, we observe a remarkable plasticity in
the central helical domain. Incoming signals induce helix rotations and
asymmetric helical bends that modify the accessible surface of the
phosphorylation site and the mobility of the ATP-binding domains, ultimately
modulating the protein’s catalytic activities.
The central four-helix bundle domain includes coiled-coil structures that
reach the histidine phosphorylation site. The trans-membrane sensor region
seems to drive the helical rearrangements. Heptad-repeat sequence features
allow for the extension or disruption of the coiled-coil towards the
N-terminus of the catalytic core, ultimately serving as a signal
transmission gear. In correlation with these movements, the flanking
ATP-binding domains, remain either rigidly fixed to the 4-helix bundle, or
otherwise free to move. We have explored the transient intradimeric
autophosphorylation state by semiflexible docking algorithms, leading to a
proposed mechanism working in trans, one monomer phosphorylating the other.
Structure-based cysteine engineering lends support to the working
hypotheses, allowing us to trap an intermediate state with disulfide bridges
between the two domains [2]. Negative cooperativity leads to phosphorylation
of only one monomer within the dimer.
Structure-based mutagenesis and protein engineering experiments in vitro and
in vivo, confirm the importance of the ‘coiled-coil’-mediated plasticity in
the conserved central phosphotransfer domain. Similar switching mechanisms
could operate in a wide range of sensor HKs. Structural studies of the
interaction of DesK with its cognate response regulator DesR are currently
underway.

[1] D. Albanesi, M. Martín, F. Trajtenberg, M. C. Mansilla, A. Haouz, P. M.
Alzari, D. de Mendoza and A. Buschiazzo, Proc Natl Acad Sci U S A 2009, 106,
16185-16190
[2] F. Trajtenberg, M. Graña, N. Ruetalo, H. Botti and A. Buschiazzo J Biol
Chem. 2010, 285, 24892-903.**