|Research directions (Projects P1-5) of our laboratory are the ones depicted in the scheme. We aim to unravel the structural dynamics of the indicated “hand-picked” biological systems that impact human health and represent highly desirable drug (antibiotic) targets. We mainly focus on the Bilobed and Ras structures.
In the scheme, a cartoon cross-section of a Gram-negative pathogenic bacterial cell (colored) is in close contact with a eukaryotic host cell (white). Ras undergoes structural dynamics signaling multiple pathways in the host. Extracellular nutrients of the host transverse the outer membrane of the bacterial cell (e.g. transferrin bound iron via the TbpA porin) and are trapped by the periplasmic (cyan) bilobed proteins and subsequently guided to the appropriate ABC transporter for uptake in the cytoplasm (orange).
We have recently shown that the bilobed proteins, synthesized at the ribosomes, with different N- (blue) or C-termini (black) are evolutionary related.
When the N-terminus is a signal peptide, the bilobed protein is transported to the periplasm by the Sec translocase. Those proteins have different asymmetric C-termini that confer distinct domain-domain dynamics that diversify the specificity and function of the bilobed structure.
Bilobed proteins with an N-terminal HTH-DNA binding domain oligomerize, lacking a C-terminus and consequently domain-domain dynamics. Instead of tertiary, the quaternary dynamics, enhanced upon binding to DNA and regulated by transcriptional effectors, dictate the function of transcription factors belonging to the LysR family.
Bilobed proteins without N-termini, have symmetrical C-termini and consequently lose the ability of domain-domain dynamics. Therefore, function by an apparent lock-and-key mechanism displaying primarily enzymatic activities.
Our experimental tools (e.g. single-molecule FRET) can probe with high spatial (Å) and temporal (ms) resolution the distance between 2 probes (D, A) placed strategically at two distinct sites on the bilobed or Ras structure. This allows to monitor in real time under physiological conditions their structural dynamics with single-molecule resolution
P1: The folding landscape of the Ras and bilobed structure
All proteins are synthesized as long, unstructured polymers that compact and fold to acquire their native 3D structure by forming a hydrophobic core. During folding, the polypeptide chain samples various conformations in the energetic landscape, to finally acquire the low-energy, more stable, native (soluble oftentimes) state. Different folding routes lead to aggregated states, like in the case of amyloid proteins causing human diseases (e.g. Alzheimer, Amyotrophic lateral sclerosis) and giving rise to the biofilms in bacteria. To diminish the risk of misfolding/aggregation, cells developed during evolution numerous types of molecular chaperones. Understanding the physical principles of folding represents a fundamental and exciting biological question and will allow to obtain a molecular understanding of protein misfolding/aggregation/amyloid formation. The Ras and bilobed structure represent ideal systems to derive basic folding principles, as those are harbored by proteins having simple and almost identical 3-dimensional structures, acting at different subcellular localizations, having completely different functionalities, and are evolutionary related.
P2: The Structural landscape of the bilobed structure: from molecular mechanisms to antibiotics and biosensors
i. A class of bilobed proteins is found exclusively in Gram-negative pathogenic bacteria (e.g. P. aeruginosa, N. gonorrhoeae; H.influenzae; M. haemolytica) and their dynamics are essential for their virulence and also affect their interactions with drugs
ii. Transferrin and lactoferrin, two key proteins for iron homeostasis and consequently critical for human health, are bilobed proteins. Understanding the means by which the two proteins bind iron so tightly and the mechanisms used for its release (in host or pathogenic cells) is fundamental for deriving a mechanistic understanding of iron homeostasis at the molecular level, but also the essential role of these proteins for the nutritional immunity
iii. Bilobed proteins are also used as (bio)sensors of toxic compounds (e.g. arsenic). We can modulate all binding properties, thus develop multiple protein-based biosensors with different sensitivities and selectivities.
P3: The Structural landscape of the Ras structure: switching Ras from undruggable to druggable
Ras is the founding member of the GTPase superfamily, sharing an identical (Ras)structure having orthologs in many organisms. Ras proteins regulate crucial cellular functions like cell survival, proliferation, differentiation and apoptosis. They are found at the tip of the signaling “pyramid”, just downstream of the cell-surface tyrosine kinase receptors (RTKs). Our aim is to elucidate the switching behavior of the structure: How are protein dynamics altered so that an identical structure yields either signaling proteins (Ras, Rho), proteins regulating vesicular transport (Rab, Arf) or even nucleoplasmic transport (Ran)?
P4. Bacterial ABC transporters: Structural dynamics during solute import
ABC transporters are ubiquitous in cells of all three domains of life and transport an enormous variety of substrates. They represent the most abundant and diverse family of transport proteins known, playing crucial roles in numerous cellular processes such as nutrient uptake, antibiotic and drug resistance, antigen-presentation, cell volume regulation and many others. Our aim is to decode the interaction of the translocator domains with the bilobed structure and elucidate salient features of the transport mechanism.
P5. The Sec translocase: Structural dynamics during protein secretion
In every living cell, approximately half of the proteome is non-cytoplasmic. The Sec pathway can handle only unfolded polypeptides that acquire their native state after they transit to the cell exterior where cleavage of their signal peptide occurs. Our aim is to obtain an exhaustive mechanistic understanding of protein secretion. The outcome of this fundamental research will have important repercussions in: i. guiding antibiotic development as the Sec translocase is essential for every bacterial life, ii. Enhancing biotech applications as optimization of protein secretion has a huge market (Medical-Clinical research, Industrial enzymes cosmetics, Food-nutrition)
Consequently, our research line is based on a 3-pronged approach:
A. Fundamental research: We are focusing on human/yeast Ras signaling and bacterial proteins. The former ones regulate many signaling pathways, being responsible for 30% of human cancers, whereas the later are responsible for bacterial transcriptional regulation (e.g. LysR transcription factors), pathogenicity (e.g. FbpA) or mediating the uptake of essential nutrients (e.g. MalE). The same (structurally) motors in vertebrates are responsible to maintain iron homeostasis (e.g. transferrin) or to regulate the concentration of neurotransmitters in synaptic clefts (ABC transporters). Inspecting those systems allows decoding fundamental biological processes: How does a bacterial motor protein work at the molecular level? How can a common structural core diversify throughout the tree of life to acquire different functionalities?
B. Drug & Biosensor development: Proteins are the major drug targets, as binding of drugs to protein clefts either abolishes an undesirable function (e.g. block FbpA dynamics, a protein essential for bacterial pathogenicity) or modulates their over-activation (e.g. Ras signaling protein activating cellular pathways). Despite the fact that dynamics of protein clefts affect drug-target interactions; such are marginally considered during drug development, as their study requires multi-disciplinarity and faces technological challenges. Importantly, the approaches we are adopting will allow to understand and modulate the binding parameters. This will permit us to regulate the specificity, selectivity and sensitivity of protein-protein and/or protein-small molecule interactions. With this knowledge, we aim to develop protein-based biosensors valuable for a plethora of applications.
C. Tech transfer / High-throughput screening: As the binding of every drug molecule to a protein cleft affects protein dynamics, we will be developing (in collaboration with pharma companies) generic high-throughput screening platforms.