Molecular Machines in Action
A fundamental property of many biological processes is that they are performed by highly organized, multicomponent macromolecular assemblies, often referred to as molecular machines. My lab studies the structural basis for assembly, regulation, and function of transmembrane molecular machines. We use a multidisciplinary approach, by combining molecular biology, genetic, cellular, biochemical, and a wide-range of structural (EM, X-ray, NMR, X-linking/mass spectrometry) tools. We are developing novel imaging and modeling technologies to visualize dynamic molecular processes in unprecedented detail in situ and in action.
Gram-negative pathogens such Yersinia, Shigella, Pseudomonas, enteropathogenic/enterohemorrhagic E. coli (EPEC/EHEC) and Salmonella are the causative agent for many diseases known to animals or humans. They range from mild to deadly outcomes and often originate as food-borne diseases. Bacterial toxins (so called effectors) are a major aspect of their pathogenicity. They are delivered via the type III secretion system (a large membrane-embedded machinery, also known as injectisome) from the bacterium to its host cell. As a consequence, translocated effector proteins have the remarkable capacity to modulate various host-cell pathways, including endocytic trafficking, gene expression, programmed cell death, or cytoskeleton dynamics that induce membrane ruffling and subsequently make the host accessible to bacterial infection.
Type III secretion system: Unfolded protein transport across membranes
Our recent structural analysis (Schraidt & Marlovits, Science 2010) of the injectisome, the most prominent, cylindrical structure of type III secretion system, revealed a potential secretion path through the central part of the membrane embedded complex. However, the inner diameter of this path is too small to accommodate a fully folded effector protein, suggesting that either the injectisome must undergo large conformational changes during transport or effector proteins need to be unfolded.
To investigate type III secretion of human pathogens, we focused (1) to determine the secretion path of injectisomes, (2) to understand the mechanism of transport, and (3) to visualize protein transport in situ. We discovered that substrates are inserted into the secretion path in a polar fashion - N-terminal regions first – and that they are transported in an unfolded state. To establish whether such behavior does in fact occur in situ, we analyzed protein transport across membranes in a near-native state by cryo electron tomography (Radics et al 2014). For the first time, we were able to visualize pathogenic type III secretion systems from Salmonella in action.
Technological development - determination of atomic structure from lower resolution cryo-EM maps
Direct electron detectors are a key aspect of the recent revolution in structural biology because they have enabled the determination of electron density maps at near atomic resolution from non-crystalline sample material, using cryo electron microscopy. However, building accurate models into these 3-5Å maps remains a challenge. We thus recently reported a new modeling approach that integrates Monte Carlo optimization with local density guided moves, Rosetta all-atom refinement, and real space B-factor fitting, yielding accurate models from experimental maps for three different systems with resolutions as low as 4.5Å (DiMaio et al Nature Methods 2015). Recently we expanded this work and developed easily used modeling tools to build accurate models at the highest possible resolution from single particle electron microscopy maps.