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Molecular Machines

Membrane-associated processes are a fundamental characteristic of all living cells. They ensure that the cells are able to effectively communicate with, and adapt to, their environment. The cells achieve this by either physically translocating molecules to the opposite site of a membrane or by receiving, transmitting, and amplifying incoming signals. Our laboratory is interested in understanding the molecular mechanism underlying such processes. Specifically, we focus on machineries capable of translocating bacterial toxins into eukaryotic cells.

Microbial Pathogenesis

One of the most exciting discoveries in the last few years is that supramolecular assemblies are one of the key elements involved in the infection of eukaryotic cells by bacterial pathogens. These systems give rise to intimate contact between cells, deliver specific toxins - which are collectively known as effectors proteins - into host cells, and possess the remarkable ability to modulate diverse regulatory networks. In particular, many Gram-negative pathogens such as Salmonella, Yersinia, Pseudomonas, or Shigella utilize the type III secretion system (TTSS) to initiate infection in eukaryotic cells. TTSS is a multicomponent system comprising more than 20 different proteins. A mere fraction of these assemble into the so-called needle complex, which is the most prominent core structure of the system. It is a membrane-associated complex with  >3.5 MDa, and is composed of a set of soluble and membrane proteins. Although it is essential for microbial infection in many animal as well as plant pathogens, the assembly of the needle complex and how the needle complex identifies and triggers efficient translocation of substrates are still poorly understood. Using Salmonella typhiumurium, we are investigating the molecular mechanisms and structural framework required to translocate effector proteins specifically and safely into eukaryotic cells.

Recent crystallographic analyses of individually separate domains, which are predicted to be located within the plasma, revealed a common structural motif organized in repeating modules. Attempts have been made to “dock” these protein domains into the needle complex structure, which resulted in several mutually incompatible locations. We have used a combination of methods including bacterial genetics, biochemistry, mass spectrometry and cryo electron microscopy/single particle analysis to experimentally determine the position of specific protein domains within the needle complex. In addition, we have identified specific sites of interaction among components of the needle complex, which are critical for stable assembly and the subsequent functional complex. Jointly, this analysis provides the first experimentally validated topographic map of different components of the needle complex of the S. typhimurium TTSS (Figures 1 and 2) (Schraidt et al., 2010).

Our topological analysis revealed that additional proteins must be present. These constitute the cup/socket structure which is located in the center of the needle complex (export apparatus). Using mass spectrometry, we were able to identify five additional candidate proteins that co-fractionate in marginal quantities with purified needle complexes. Subsequent structural analysis revealed the absence of the cup/socket, suggesting that one or more of these proteins is required to build up the cup/socket (Figure 3a). We were also able to show that these proteins nucleate the coordinated assembly of the needle complex (Wagner et al., 2010).

Efficient effector protein translocation is known to occur only after host cell contact. Therefore, it is conceivable that the extracellular filament is a key player in the transmission of this information, probably due to small conformational changes throughout the filament. This hypothesis is supported by mutations found in the homologous Shigella needle filament, which convert the system into a constitutively “on” state. If this is true, it would be justifiable to presume that the filament is provided with a certain degree of structural heterogeneity in order to accommodate the required conformational plasticity for signal transmission. Therefore, we analyzed the structure of the needle filament by cryo electron microscopy (Figure 3B) and discovered that the structure is, indeed, highly variable (Galkin et al., 2010).

Although the design of the TTSS appears to be conceptually simple, many questions remain unanswered: How dynamic is the entire assembly process? How are substrates recognized by the needle complex? What is the molecular mechanism of protein translocation? We have begun to address some of these questions. By understanding the molecular mechanism of TTSS-mediated protein transport, we hope to provide a basis for the development of novel therapeutic strategies that will either inhibit its activity or modify the system for targeted drug delivery.

Molecular Machines Fig. 1
Figure 1 (click to view legend)

Figure 1: The needle complex is the core structure of the type III secretion system. (A) Needle-like structures (approx. 50 nm) that extend into the extracellular environment are visible on the surface of osmotically shocked S. typhimurium. (B) Isolated complexes from wild-type and a invG mutant strain are structurally different. (C) Isolated needle complexes harboring poly-histidine tags at various positions in PrgH or PrgK can be specifically labeled using nanogold.



Molecular Machines Fig. 2
Figure 2 (click to view legend)

Figure 2: Organization of PrgH, PrgK and InvG within the needle complex (A) End view class average derived from single particle analysis from negatively stained electron microscopy images of sub-structures of the inner rings. The sub-structures were obtained by selective disassembly of needle complexes isolated from a mutant strain encoding for a C-terminally, four amino acid truncated PrgH. Rotational cross-correlation analysis revealed that the maximum of the cross-correlation peak is repeatedly obtained every 15°, demonstrating that the larger concentric rings of the inner ring structure exhibit 24-fold symmetry. (B) Proximity of specific domains of the base proteins, InvG, PrgH, and PrgK within the needle complex. The block diagrams shows the three major base proteins, InvG, PrgH, and PrgK, and covalent cross-links of peptides obtained from chemically derivatized needle complexes at primary amino groups. (C) Topographic model of the needle complex: Localization of domains of InvG, PrgH, and PrgH within the base of the needle complex. The N-terminal domain of InvG (blue-grey) reaches far down into the neck region and is in close contact with the C-terminal domain of PrgH (white and grey). Sites of interaction found by cross-linking and mass spectrometry are labeled as red dots. The N-terminal domain of PrgH is pointing to the cytoplasmic side of the complex, and interacts with the C-terminal domain of PrgK. For both, no high-resolution structure is available as of now. The N-terminal domain of PrgK is located within the complex and is therefore packed into its position by PrgH from the side and InvG from the top.



Figure 3 (click to view legend)

Figure 3: Sub-structures of the needle complex. (A) Structural analysis of complexes from strains lacking the export apparatus proteins (SpaSPQR, InvA) reveal marked differences in the central portion of the complex (socket/cup) compared to the wild type. (B) The needle filament protein PrgI is the building block of the extracellular needle filament.