Structural and functional characterization of archaeal membrane proteins

Structural characterization of the mechanosensitive channel of small conductance MscCG from C. glutamicum

Structural Investigations of the Stressosome Complex

Structural and functional characterization of eukaryotic osmolyte transporters

Structural investigation of S-layers from Pyrobaculum aerophilum



Structural and functional characterization of archaeal membrane proteins

Our research focus on the structural characterization of all membrane proteins in the host-symbiont system Ignicoccus hospitalis-Nanoarchaeum equitans, which are suggested as putative archaeal ancestor of mitochondria. Here, our group is working in close collaboration with the Archaea Centre and the group of Harald Huber. Ignicoccus hospitalis is an anaerobic obligate chemolithoautotrophic Crenarcheaon that exhibits beside other highly unusual features an exceptional cell compartment formation, which is characteristic for eukaryotes. Two membranes are forming an intermembrane compartment (IMC) of a variable width of 20 to 1000 nm filled with membrane vesicles. The energized “outer cellular membrane” (OCM) comprises an ATPase and a H2:sulfur oxidoreductase complex. Moreover, pore complexes of 7 and 24 nm, respectively, were observed in electron micrographs (2). By crystallizing and functionally characterizing proteins located in either of the two membranes and in the vesicles we want to learn about the evolution of eukaryotic transport and protein translocation systems.

In our group we clone, express and crystallize proteins originated from these different membranes to gain insights into their composition, distribution, and function. The final goal will be to understand the unique role of I. hospitalis  and its symbiont Nanoarchaeum equitans as putative archaeal host system in the evolution of mitochondria.


References:

1. H. Huber et al., (2012) The unusual cell biology of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. Antonie Van Leeuwenhoek, 102(2), 203-219.

2. T. Burghardt et al., (2007) The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex. Mol Microbiol. 2007, 63(1), 166-76.

 

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Crystals of a membrane bound
complex of Ignicoccus hospitalis


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Localization of membrane proteins
in Ignicoccus hospitalis






Structural characterization of the mechanosensitive channel of small conductance MscCG from C. glutamicum

Prokaryotic mechanosensitive channels (MS) play a major role in the cellular protection system under hypo-osmotic stress situations. The membrane protein MscCG from Corynebacterium glutamicum was previously described as a mechanosensitive channel of small conductance. These MS-channels act as emergency valves upon sudden osmotic downshift when excessive water inflow increases the turgor pressure. They sense the increased membrane tension and get activated within milliseconds to release compatible solutes to the environment and prevent the cell from lysis. Recently, MscCG was shown to be involved in the glutamate efflux of C. glutamicum under glutamate production conditions like the cell- growth under biotin limitation or the treatment with penicillin. Interestingly, MscCG was also reported to function under hyper-osmotic stress conditions - in fine-tuning the steady-state concentration of the compatible solute betaine in the cell.

MscCG from C. glutamicum (533 aa) exhibits 23% sequence identity to the MscS-channel (mechanosensitive channel of small conductance) from E. coli (286 aa), which forms a functional homoheptamer. The most similarities between MscCG and MscS are in the N-terminal part (see homology model) which represents the channel domain. Furthermore MscCG contains a long C-terminal domain exhibiting a unique sequence. This C-terminal part was recently shown to carry an additional transmembrane domain.

Therefore our group is interested in the structural differences of MscCG to other MscS-channels and we want to learn more about the so far unknown C-terminal domain of MscCG.




References MscCG:

1. Ruffert et al., (1999) Identification of mechanosensitive ion channels in the cytoplasmic membrane of Corynebacterium glutamicum. J Bacteriol, 181, 1673-1676.

2. Nakamura et al., (2007) Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production. Appl Environ Microbiol,  73, 4491-4498.

3. Bass et al., (2002) Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science, 298, 1582-1587.

4. Börngen et al., (2010) The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaption. Biochimica et Biophysica Acta, 1798, 2141-2149.

5. M. Becker, et al., (2013) Glutamate efflux mediated by Corynebacterium glutamicum MscCG, Escherichia coli MscS, and their derivatives. Biochim. Biophys. Acta, http://dx.doi.org/10.1016/j.bbamem.2013.01.001

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Homology model. The monomeric structure model  of the truncated MscCG∆247 (blue) in comparison to the E.coli MscS-monomer structure (2oau)(yellow).






Structural Investigations of the Stressosome Complex


Bacteria have almost permanently to deal with conditions which provide physical stress. Thus it is essential for bacterial cells to be able to detect and counteract a wide range of environmental stress such as heat, salt, toxins, UV-light and ethanol. One adaption to such circumstances is the general stress response (GSR) of Bacillus subtilis that permits the activation of the sigB factor which binds the RNA polymerase for transcription of 150 general stress genes to protect the cell. This GSR in B. subtilis is mediated by a 1.8 MDa cytoplasmic stressosome complex - the key signaling system which is composed of multiple copies of three different proteins (RsR, RsbS and RsbT).

Due to the fact that in representatives of most bacterial phyla gene clusters which encode for stressosome homologs are present, this concept of a stressosome complex is presumably more often distributed among bacteria. The stressosome itself plays therefore an important role in many different kinds of environmental stress regulations. Up to now it is not known how different stress signals are detected and mediated by the stressosome. Our research is focused on the stressosome complex. Therefore we express and purify the protein complex and investigate the stressosome via electron microscopy (A) and 3D reconstruction (B).

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A: EM negative stain
B: few class-sums (program EMAN2)





Structural and functional characterization of eukaryotic osmolyte transporters

Secondary transporters play important roles in maintaining vital functions in the mammalian kidney, which constantly encounters major changes in osmolality during urinary concentration. The renal hydration state is adjusted by accumulation of several osmolytes, one of which is betaine. Betaine-mediated volume regulation prevents severe dysfunction of the kidney and the central nervous system. Moreover, betaine counteracts the destabilizing action of urea on protein structures in renal cells.

Renal betaine uptake is facilitated by BGT-1 (GAT2, gene name SLC6A12), which belongs to the osmolyte branch of the human NSS or Solute Carrier 6 (SLC6) family.  Besides kidneys, BGT-1 is found in the brain and in the liver mediating also the transport of the neurotransmitter GABA. Both betaine and GABA transport is chloride-dependent and coupled to the transport of 3 sodium ions. During hypertonicity, BGT-1 transcription and insertion to the basolateral plasma membranes is increased and transport of betaine is up-regulated. However, the molecular mechanism of sensing hypertonicity and subsequent regulatory membrane insertion of BGT-1 is unknown. Controlled folding by N-glycosylation might be one regulatory mechanism, as the interaction with N-glycans affects targeting and functional insertion to the plasma membrane in many different proteins.

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Structural investigation of S-layers from Pyrobaculum aerophilum


Surface (S)-layers form the outermost cell compartment of many bacteria and archaea. They consist of monomolecular arrays of protein or glycoprotein species, maintain structural rigidity and protect the organism from environmental elements. Their 2D crystalline lattice shows either an oblique, tetragonal or hexagonal symmetry the latter being predominantly found in archaea.

In our group we focus on structural investigation of S-layers from archaea using electron microscopy, especially electron crystallography and electron tomography.

 
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