spEakers
Hashim Al-Hashimi
Columbia University, New York, USA
Bringing nucleic acid structures to life using NMR
If we deeply understood how biomolecules interacted with one another, there would be nothing left to learn in biology because every biological phenomenon is ultimately the product of biomolecular interactions. To date, much effort has focused on determining the three-dimensional structures of biomolecules and their complexes at atomic resolution. However, despite seven decades of the structure-function paradigm, we still haven’t attained a predictive understanding of even simple biochemical reactions, and we are far from being able to control them rationally. In solution, biomolecules do not fold into a single structure; instead, form dynamic ensembles of thousands of different conformational states, each forming with a specific probability and lifetime. This probability distribution of conformations changes when biomolecules bind to partner molecules, thus activating new functions. In this lecture, I will describe how advances in solution-state NMR spectroscopy are making it possible determine conformational ensembles of RNA and DNA molecules at atomic resolution and over timescale ranging from picoseconds to seconds. I will discuss how dynamic ensembles have been successfully used to build quantitative and predictive models for several biochemical reactions, including DNA replication fidelity, RNA folding, RNA-ligand and RNA-protein binding, and hybridization kinetics. These and other studies mark the beginnings of a dynamics revolution in structural biology which promises a deeper, more quantitative, and predictive understanding of life processes.
Lindsay Baker
University of Oxford, UK
Combining NMR and cryoET for ‘in situ’ structural biology
NMR spectroscopy and electron cryotomography (cryoET) are highly complementary techniques, spanning different scales in both space and time that are important for biological function. In this talk, I will discuss why combining NMR and cryoET provides a powerful platform for studying complex molecular systems. In particular, I will focus on how they allow us to study membrane proteins in native membranes, as well as compartmentalisation and protein-protein interactions.
Marc Baldus
Utrecht University, The Netherlands
Ultra-high field NMR for in-situ life science research & beyond
Increasing evidence suggests that a full understanding of biomolecular function and disease requires in-situ approaches that probe molecular structure and dynamics in a native setting. Advancements in NMR instrumentation have been critical for enhancing the use of NMR spectroscopy to study biomolecular systems in such conditions. In our contribution, we will discuss high-end NMR applications to study complex biomolecular systems in a native cell setting. These methods maximize spectral resolution and sensitivity and are geared towards elucidating the dynamic landscape of proteins and other biomolecules in bacterial, fungal and human cells and cell organelles.
Andrew Baldwin
University of Oxford, UK
Title to be confirmed
Orsolya Barabas
University of Geneva, Switzerland
Structural insights into the transposition of
antibiotic resistance
Conjugative transposons drive horizontal gene transfer and promote the spread of antibiotic resistance genes. They can move across a wide range of bacteria and their movements have been linked to the emergence of multidrug-resistant pathogens, which present a major public health challenge world-wide. Yet, their molecular mechanisms are unclear.
Using a dedicated computational pipeline, we identified the most wide-spread conjugative transposons and characterized their diversity, genetic cargo, and transfer dynamics. Focusing on a broadly distributed element from Gram-negative pathogens, we delineated the biochemical pathway and determined high-resolution crystal and cryo-EM structures of the multicomponent protein-DNA assemblies involved in its movement. The structures capture various steps of transposition and reveal an intricate interplay between the core transposition machinery and host-encoded proteins. Protein interactions fold transposon and genomic DNA in unique ways to cut, exchange and re-join DNA strands in a tightly regulated manner. Asymmetric DNA recognition, directed assembly and specific cleavage mechanisms facilitate transposition irrespective of the genomic insertion site, expanding gene transfer across species.
These results shed new light onto the molecular strategies of conjugative transposons and show how they evolved to effectively spread genetic traits, opening new possibilities to block transposition and control the spread of antibiotic resistance.
Martin Blackledge
Institute of Structural Biology, Grenoble, France
NMR Provides Unique Insight into the Functional Dynamics and Interactions of Intrinsically Disordered Proteins involved in Viral Replication
Proteins are inherently dynamic,
exhibiting conformational freedom on many timescales, implicating structural
rearrangements that play a major role in molecular interaction, thermodynamic
stability and biological function. Intrinsically disordered proteins (IDPs)
represent extreme examples where flexibility defines molecular function. In
spite of the ubiquitous presence of IDPs throughout biology, the molecular
mechanisms regulating their interactions remain poorly understood. We use NMR
spectroscopy to develop a unified description of the dynamics of IDPs as a
function of environmental conditions, from membraneless organelles to in-cell,
and to map these complex molecular recognition trajectories at atomic
resolution, from the highly dynamic free-state equilibrium to the bound state
ensemble.
Examples include the nuclear pore,
where weak interactions between the nuclear transporter and highly flexible
chains containing multiple ultra-short recognition motifs, facilitate fast
passage into the nucleus, the replication machinery of Measles virus, where we
use NMR to characterize the 92 kDa complex formed between the highly disordered
phosphoprotein and the nucleoprotein prior to nucleocapsid assembly – a process
that we can also follow in real-time. These proteins undergo liquid-liquid phase
separation upon mixing and we can combine NMR and fluorescence to describe the
molecular basis and functional advantages of this phenomenon. NMR also sheds
new light on the molecular basis of host adaptation of influenza polymerase,
via a highly dynamic interaction, and reveals the dynamic assembly of
SARS-CoV-2 nucleoprotein with its viral partner nsp3.
Davide Calebiro
University of Birmingham, UK
GPCR signaling in space and time: It’s all about dynamics
Whereas structural studies have provided major insights into the activation mechanisms of G protein-coupled receptors (GPCRs), how these receptors operate in our cells to produce specific biological responses remains insufficiently understood. My group develops innovative optical methods based on FRET/BRET and single-molecule microscopy that allow us studying GPCR signalling in living cells with unprecedented spatiotemporal resolution. Using these approaches, we were among the first to demonstrate that GPCRs are not only active at the plasma membrane but also at intracellular sites, which has challenged the classical model of GPCR signalling. Ongoing work in our lab is dedicated to further clarifying the physiological and pharmacological implications of GPCR signalling at intracellular sites, with a particular focus on metabolically relevant GPCRs. In parallel, we have further developed our single-molecule approaches, which we previously used to investigate receptor-G protein interactions, to study other fundamental aspects of GPCR signalling such as the mechanisms involved in b-arrestin recruitment and activation. Altogether, our findings reveal that the molecular events governing GPCR signalling in our cells are much more complex and dynamic than previously thought, which has important implications for our understanding of GPCR signalling and the future development of more effective and better tolerated drugs targeting this important family of membrane receptors.
Teresa Carlomagno
University of Birmingham, UK
An integrative structural biology approach to understand functional mechanisms of disordered regions in biological complexes
After >60 years of structural biology, the complexity of both the
molecules we study and the questions we pose has increased enormously. Magnetic
resonance makes vital contributions across biological and medical fields, due
to its unique ability to study molecules in both the spatial and temporal
dimensions, reveal minorly-populated conformations and cope with structural
disorder. Furthermore, methyl TROSY and 15N,13C-direct-detection
experiments allow molecular species as large as 1 MDa to be interrogated by
solution NMR.
In our laboratory, we use and develop integrative structural biology
approaches that employ NMR together with complementary methods to understand
the functional mechanism of high-molecular-weight complexes with enzymatic
activity. Integrative structural biology in solution is particularly relevant
for enzymes that contain disordered parts or for transiently forming complexes,
where X-ray crystallography or electron microscopy fail.
I will demonstrate the approach on the example of a
multi-protein complex responsible for acetylation of histone H3. The complex
consists of the fungal histone acetyltransferase Rtt109, the histone chaperones Asf1 and Vps75 and the H3:H4 histone dimer. Rtt109 is required for histone H3 K9, K27 and K56 acetylation in fungi
and is thus an attractive drug target in the fight of fungal infections. Rtt109 activity requires two structurally unrelated histone chaperones, Asf1
and Vps75. These proteins activate Rtt109 via different mechanisms, with Rtt109 –Asf1 association being
necessary for K56 acetylation, and Rtt109–Vps75 association
being required for K9 acetylation.
Using our
integrative structural biology approach, we explain how Vps75 promotes
acetylation of residues in the H3 N-terminal tail: the chaperone engages the
disordered H3 tail in fuzzy electrostatic interactions with its own disordered
C-terminal domain and thereby confines the H3 tail to a wide cavity faced by
the Rtt109 active site. These fuzzy interactions between disordered domains
achieve localization of the H3 tail to the catalytic site with minimal loss of
entropy, and may represent a universal mechanism for substrate localization in
enzymatic reactions involving long, highly-disordered substrates.
John Christodoulou
University College London, UK
The ribosome lowers the entropic barrier of protein folding
Most proteins fold co-translationally during biosynthesis on the ribosome and co-translational folding energetics, pathways, and outcomes of many proteins have been found to differ considerably from those in refolding studies. The origin of this folding modulation by the ribosome has remained elusive. We have determined structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome entropically destabilises the unfolded nascent chain. Quantitative 19F NMR shows that this destabilisation reduces the entropic penalty of folding by up to 30 kcal.mol-1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains. The entropic effects also result in the ribosome protecting the nascent chain from mutation-induced unfolding, suggesting a crucial role of the ribosome in supporting protein evolution. Our findings thus establish the physical basis of the distinct thermodynamics of co- translational protein folding.
Clemens Glaubitz
University of Frankfurt, Germany
Catalytic and allosteric phenomena in transmembrane receptors and transporters revealed by solid-state NMR spectroscopy
ABC transporters play an important role in various
cellular processes. They have a similar architecture and a common mechanism
that combines ATP hydrolysis with substrate transport. MsbA is located in the
inner membrane of Gram-negative bacteria and functions as a floppase for the
lipopolysaccharide (LPS) precursor core LPS, which is involved in the
biogenesis of the bacterial outer membrane. It is part of a larger LPS
transport machinery that also includes the LPT system, which maintains the
double membrane architecture and therefore plays a role in antibiotic
resistance. Fundamental aspects of the LPS transport mechanism and its
energetic coupling remain to be elucidated. Based on solid-state NMR, we have
uncovered new aspects of the catalytic mechanism of MsbA, allosteric
interactions between the transmembrane and nucleotide-binding domains and
gained first insights into the conformational dynamics of LptB2FG, the ABC
transporter that drives the LPT system and connects the inner and outer E.
coli membrane.
Another prime example of the use of solid-state NMR spectroscopy are
photoreceptors and in particular microbial rhodopsins. This protein family has
experienced a renaissance in recent years due to the discovery of its frequent
occurrence. It is striking that a similar protein architecture forms the
framework for a multitude of different functions (light-controlled ion pumps,
ion channels, sensors). The elucidation of their functional mechanisms requires
experimental and computational approaches down to the quantum chemical level. Here, solid-state NMR and in particular the possibilities of increasing
sensitivity through dynamic nuclear polarisation offer new possibilities, which
are discussed for the case of the light-induced and cryo-trapped photointermediates
of these photoreceptors.
Flemming Hansen
University College London, UK
Developing artificial intelligence to propel ultra-high field NMR into the future
Artificial intelligence (AI) and deep learning are now established as some of the most important technologies of our time. The uptake of AI in nuclear magnetic resonance (NMR) spectroscopy has until recently been slow - but this picture is now swiftly changing.
It will be shown how deep neural networks (DNNs) can be trained to transform dramatically improve the resolution of complex NMR spectra. Specifically, NMR spectra are often complicated by homonuclear scalar couplings and broad signals. Our recent work shows that virtual decoupling and sharpening of signals can confidently be achieved by appropriately trained DNNs. Another area, where we have focussed our developments is for autonomous analysis of complex NMR data. Analyses of NMR data often hinge on complex least-squares fitting procedures and human intuition. Deep neural networks will be presented for the analysis of chemical exchange saturation transfer data, where the DNN not only accurately predicts the relevant parameters, autonomously, but it also determines the uncertainties associated with these predictions.
All the DNNs developed do not contain any parameters for the end-user to adjust and the methods therefore allows for truly autonomous analysis of complex NMR data.
Gabriella Heller
University College London, UK
Picosecond Dynamics of a Small Molecule in Its Bound State with an Intrinsically Disordered Protein
Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules. Currently, only a few instances are known where small molecules have been observed to interact with IDPs, and this situation is further exacerbated by the limited sensitivity of experimental techniques to detect such binding events. Using experimental nuclear magnetic resonance (NMR) spectroscopy 19F transverse spin-relaxation measurements, we discovered that a small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 ± 110 μM. Our analysis also allowed us to determine the rotational correlation times (τc) for the free and bound states of 5-fluoroindole. In the free state, we observed a rotational correlation time of 27.0 ± 1.3 ps, whereas in the bound state, τc only increased to 46 ± 10 ps. Our findings imply that it is possible for small molecules to engage with IDPs in exceptionally dynamic ways, in sharp contrast to the rigid binding modes typically exhibited when small molecules bind to well-defined binding pockets within structured proteins.
Babis Kalodimos
St. Jude Children's Research Hospital, Memphis, TN, USA
The Conformational Landscape of Protein Kinases
Protein kinases regulate almost every aspect of cellular function. Changes in the expression, localization in the cell, mutations or chromosomal rearrangements of kinases can cause a number of cancers and other diseases. Cancer ‘driver’ mutations occur very frequently in kinase genes. In fact, the kinase domain is the domain most frequently encoded by cancer genes. Tremendous progress has been made in understanding the structure, function, and mechanisms of regulation of protein kinases. However, it has proved challenging to monitor these transitions and structurally characterize the manifold of conformational states inherently populated by a kinase. In the absence of such information, the mechanisms underpinning the response of kinases to physiological and pathological processes remain poorly understood. I will discuss how we structurally and energetically dissect the mechanisms underpinning the function and operation of a number of important protein kinases. We elucidate regulatory and drug-resistance mechanisms as well as how key structural elements and motifs control the activation/inhibition processes in kinases.
Józef Lewandowski
University of Warwick, UK
Applications
of solution and solid-state NMR in studying biosynthesis and modes of action of
natural products
In
this presentation we discuss a few examples of how solution and solid-state NMR
can be leveraged in the context of integrated structural biology of systems
involved in biosynthesis of natural products, e.g. antibiotics, and the natural
products themselves. We will highlight pros and cons of the two approaches in
different scenarios, some opportunities and limitations. We will explore
complementarity of NMR with other approaches, e.g. LC-MS based carbene
footprinting and molecular dynamics simulations.
Steve Matthews
Imperial College London, UK
Integrated structural biology of Substrate and Antibody recognition by Group A streptococcal anti-chemotactic proteases
Streptococcus pyogenes, also known as group A streptococci (GAS), is a pathogenic bacterium contributing to more than half a million deaths globally each year. It is a major cause of invasive infections. Additionally, it can lead to rheumatic heart disease, an autoimmune condition resulting from recurrent streptococcal infections, especially throat infection. To survive, this bacterium uses two enzymes, SpyCEP and ScpA, to prevent the immune system white blood cells (neutrophils) from being recruited to the infection site. SpyCEP and ScpA are cell surface serine proteases (CEPs) which enzymatically cleave critical regions from host proteins that activate the immune system, thereby disabling them. SpyCEP targets and degrades the entire family of CXC immune signalling molecules, while ScpA dismantles C5a, as well as C3a. Despite their importance and being key players in developing a multicomponent vaccine, there is striking lack of a detailed understanding of how these proteins function. Current models are also insufficient to define their precise mechanisms of action. This lecture will highlight how structural and biochemical techniques are uncovering the mechanisms how these CEPs disarm immune system components and are recognised by the host antibody response.
Rina Rosenzweig
Weizmann Institute of Science, Israel
Molecular Chaperones in Health and Disease - What we can Learn by NMR
Molecular chaperones are a diverse group of proteins vital to
defending cells from the dangers of protein misfolding and aggregation,
phenomena implicated in a host of debilitating human conditions, such as
Alzheimer’s, Parkinson’s and Huntington’s diseases, as well as many
neuromuscular disorders. Chaperones perform this protective function by
facilitating proper protein folding and assembly, refolding misfolded proteins,
preventing and even reversing aggregation, and delivering irreparably
damaged proteins for disposal. It is therefore not surprising that genetic
mutations in chaperone proteins are implicated in multiple human disorders,
including neurodegeneration, myopathies, and even cancer. Due to their large
size and dynamic nature, though, little is known regarding how such mutations
affect chaperones’ functions, leading to disease.
Here we
demonstrate how a combination of methyl-TROSY NMR experiments and dynamic
CPMG-RD and relaxation measurements can unveil the mechanism of function of
chaperones, thus deciphering the core of their malfunction in disease. For
example, using Methyl-TROSY NMR we show that pathogenic mutants of the 1MDa
DNAJB6 differ structurally from the WT chaperone, allowing the unregulated
recruitment and hyperactivation of Hsp70, depleting its levels in cells and
causing Limb Girdle Muscular Dystrophy.
Michael Sattler
Technical University Munich, Germany
NMR and integrative structural biology to study dynamic molecular interactions - from molecular chaperones to splicing regulation
We combine solution NMR, X-ray crystallography, small angle scattering, and cryo-EM in integrative structural biology to study multidomain (RNA-binding) proteins and protein complexes that play important roles in RNA-based gene regulation, cellular signalling and molecular chaperones. NMR is an important, versatile method to study molecular interactions and conformational dynamics of protein, RNAs and their complexes and for structure determination of small and dynamic complexes, which are difficult to study by crystallography and cryo-EM. NMR methods can be readily employed for validation of structural models predicted by deep learning methods, such as AlphaFold2.
The lecture will highlight NMR approaches available for such studies, providing examples from recent applications in our lab that highlight dynamic protein-RNA interactions, RNA structure and dynamic conformational states in molecular chaperones and benefits of ultrahigh-magnetic fields at 1.2 GHz proton Larmor frequency.
Ben Schuler
University of Zurich, Switzerland
Probing the interaction dynamics of disordered proteins: From disordered complexes to phase separation
The functions of proteins have traditionally been linked to their folded structures, but many proteins perform essential functions without being folded. These intrinsically disordered proteins (IDPs) are not only characterized by highly dynamic conformational ensembles, but they exhibit unconventional interaction mechanisms that are important for their functions. I will focus on highly charged IDPs and illustrate how single-molecule spectroscopy combined with simulations and other methods can be used to probe their dynamics, interactions, and phase separation.
Remco Sprangers
University of Regensburg, Germany
Beyond
static structures: quantitative dynamics in the
500 kDa eukaryotic RNA exosome complex
Molecular
machines play pivotal roles in all biological
processes. Most structural methods, however, are unable to directly probe
molecular motions. We demonstrate that dedicated NMR experiments can
provide quantitative insights into functionally important dynamic regions
in very large asymmetric protein complexes. We establish this for the 410
kDa eukaryotic RNA exosome complex that contains ten distinct protein
chains. NMR data reveal site-specific interactions and conformational
changes in regions that are invisible in static cryo-EM and crystal
structures. In particular, we identify a flexible plug region that blocks
an aberrant route of the RNA to the active site. Our work thus
demonstrates that a combination of state-of-the-art structural biology
methods can provide quantitative insights into molecular machines that go
well beyond static images.