NMR spectroscopy for the elucidation of conformation and communication networks within and between proteins and nucleic acids
Recent breakthroughs in nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and other biophysical techniques have reshaped our views of protein dynamics and function. We are beginning to grasp a more holistic and fascinating reality in which many, if not the majority of the proteins and nucleic acids are operative as communication hubs that are interconnected with many others. Domain architecture is defined by the 3D average structure, but biomolecules are inherently dynamic systems. Our dynamic picture is still largely incomplete because no adequate methods are available to detect concerted conformational transitions at atomic resolution. These transitions, however, form the structural basis for relaying information from one molecular site to another (so-called allostery).
NMR spectroscopy is one of the principal techniques to answer fundamental questions addressing protein/nucleic acid function due to its analytical potential at an atomic-level. Using NMR, we contribute to the understanding of communication networks by modeling realistic ensembles of structures, identification of allosteric mechanisms, studying folding and interactions with other proteins, nucleic acids or small ligands. An important pillar of this program is the establishment of our recently developed exact nuclear Overhauser enhancement (eNOE) methodology for a realistic representation of molecular spatial sampling as a standard tool in the NMR/structural biology community.
Standard structure determination by NMR spectroscopy makes use of the abundant number of experimentally readily accessible NOE rates. These rates are typically employed in a semi-quantitative manner because the measurement is obscured by spin diffusion, low signal-to-noise ratio and technical limitations. We have recently proposed a novel method for the exact measurement of NOEs (eNOEs). Distances obtained from eNOEs can have an experimental error of only ~ 0.1 Å. The collection of thousands of eNOEs throughout a biomacromolecule serves as an excellent probe towards a more complete representation of the structural landscape.
Motor adaptors in complex dynamic protein assemblies associated with the motor cytoplasmic dynein
Cytoplasmic dynein 1 (‘dynein’), a multi-protein complex of 1.2 megadaltons, is the predominant microtubule minus-end-directed motor in animals and humans. Dynein participates in a wide range of cellular activities, ranging from the transport of proteins, RNA, and vesicles to nuclear migration and cell division. Specific adapter proteins link dynein to cargo and activate the motor by forming a ternary complex with dynein and its essential co-activator dynactin. Mutations in dynein, as well as in its co-factors and adapters, have been linked to multiple neurodegenerative diseases. Several adapters have been shown to interact with the C-terminal tail of dynein light intermediate chain (LIC), which is predicted to be disordered, and mutations in the interacting segments have been implicated in spinal muscular atrophy.
It is our aim to use NMR-based experiments with the LIC C-terminal tail to provide molecular insight into how the elongated, flexible scaffold engages with functionally diverse dynein adapters. This project is realized in collaboration with the group of Dr. R. Gassmann at the Institute for Molecular and Cell Biology (IBMC) in Porto.
Structural landscape of Pin1 allostery and WW domain folding
The human peptidyl-prolyl cis/trans isomerase (PPIase) Pin1 exhibits an intricate example of allostery in a two-domain protein with high biological significance. It is involved in the regulation of mitosis, protection against Alzheimer’s disease, increase of hepatitis C infection and it is overexpressed in many human cancer cells. The catalytic domain isomerizes pS/T-P containing peptide motifs. Not only the structure but also the dynamics of the native 34-residue WW-domain seems to be responsible for the specificity of binding. Interestingly, the WW domain transmits information from its binding site to the spatially separated catalytic PPI site. The underlying mechanism remains a riddle. In our initial studies of the apoform WW domain, we identified a dynamic network spanning most of the mini core and loop 2.
It is our aim to get an atomic-resolution view of the allosteric functionality of the entire Pin1, and the folding pathway(s) of its WW domain.
Long-range coupling networks in PDZ domains
PDZ (post-synaptic density-95/discs large/zonula occludens-1) domains are highly abundant modules that mediate protein-protein interactions in eukaryotes such as organizing signaling pathways. They have been proposed to exhibit long-range evolutionary, energetic, structural and dynamical intra-domain couplings that link distal molecular docking sites within the domain. However, these results have been mutually challenged by the according studies. A consensus picture is slowly emerging as the studies use different approaches and various members of the PDZ family, which may in part deviate from the canonical characteristics. In a broader context, PDZ domains represent one of the first case studies on allostery mediated through dynamics.
Aim. We apply the eNOE-and standard NMR protocils to the second PDZ domain of a tyrosine phosphatase in free and peptide-bound form. Our conformational networks will then be related to the various proposed networks. An NMR-based evidence of the presence of correlated motion along the proposed pathways would constitute a major advance both in NMR methodology, as well as towards an understanding of dynamics-function relationships and allostery.
Allostery in protein-RNA interaction
Non-coding RNA (ncRNA) has recently generated much interest due to its ability to sequester protein. In the bacterial Csr/Rsm system, which is the most general global post-transcriptional regulatory system responsible for bacterial virulence, mRNA such as CsrB or RsmZ initiates translation through sequestration of homodimeric CsrA-type proteins bound to ribosome-binding sites of messenger RNAs.
Our collaborator F. Allain (ETH Zurich) has shown that in the case of Pseudomonas fluorescens, RsmE protein dimers assemble sequentially and specifically onto ribonucleoprotein structures, when binding the non-coding RNA RsmZ. The binding cascade of up to five RsmE dimers to one RsmZ is cooperative but binding of the second stem-loop (SL) happens via a negative allosteric effect. Our preliminary NMR analysis of the binding of two isolated SL2 to RsmE suggests a mechanism of inter-subunit allostery in RsmE that involves two distinct intermediate states in slow exchange.
It is our aim to reveal the allosteric mechanism underlying SL2 binding to RsmE at atomic level using eNOE-based and established NMR techniques. On a more fundamental level, the elucidation of this allosteric mechanism may uncover more general principles on how RNA binding domains in tandem or homodimeric RNA binding proteins fine tune their RNA binding affinity.