New technologies, tools, and approaches, often spanning several disciplines, are revolutionising biology and providing unprecedented opportunities to advance the frontiers of bioscience knowledge. Fascinated by the power of new technologies on the progress of science, Jan Steyaert focusses on the development of a generic molecular toolbox to study the conformational changes in proteins. We knew that many proteins are flexible and that their function is tightly connected to conformational changes but for many years, these different conformations were intractable for structure determination by any general method.
‘Freezing’ proteins to study their structure
The Steyaert lab pioneered the use of Nanobodies as exquisite tools to freeze dynamic proteins into single functional conformations. Nanobodies are the variable domains of heavy-chain only antibodies that naturally occur in camelids. X-ray crystallography or single particle cryo-EM can then be used to determine the structures of different stills of the same moving biomolecule. The power of this approach compares to the series of photographs that Eadweard Muybridge made in 1878 to uncover if a horse that trots or gallops does ever become fully airborne.
Expanding the Nanobodies’ utility
Jan Steyaert discovered nanobodies to study the dynamics of the highest hanging fruits in structural biology including amyloidogenic proteins, membrane proteins and transient multiprotein complexes. And he is expanding the utility of such conformational Nanobodies for integrative structural biology. Conformational Nanobodies can be introduced inside living cells (Intrabodies) as conformational biosensors. His lab uses protein engineering to make such Nanobodies amenable to single particle cryo-EM (Megabodies). The Steyaert lab also extensively explored the benefit of locking targets in drugable conformations for better-focused drug discovery (Confobodies).
Most of this work is performed in collaboration with top scientists worldwide, aiming to validate his toolbox on their most challenging projects. Jan Steyaert also (co)founded 3 companies (Ablynx, Agrosavfe and Confo Therapeutics), providing a living example of how basic research can be translated into value for the society.
Pioneering Nanobody-assisted X-ray crystallography
Jan Steyaert started his scientific career as a structural enzymologist. My expertise stretches from protein engineering and (pre-) steady state kinetics to protein expression and purification, biophysics, structural biology and display techniques. Around 2008, I transferred my skills to a new field at the interface of structural biology and antibody technology. Most recently, my lab pioneered Nanobody-assisted X-ray crystallography as a leading-edge technology, enabling the investigation of the highest hanging fruits of structural biology including membrane proteins, amyloidogenic proteins, and multiprotein complexes.
Locking agonist-bound active state GPCR conformational states.
The active-state conformations of GPCRs are unstable in the absence of specific cytosolic signalling partners representing key challenges for structural biology. In collaboration with Brian Kobilka, we generated Nanobodies against the β2 adrenergic receptor (β2AR), the muscarinic acetylcholine receptor (M2R) and the μ-opioid receptor (MOR) that exhibit G protein-like behaviour, and obtained agonist-bound, active-state crystal structures of receptor●Nb complexes of β2AR, M2R & MOR amongst others.
Nanobodies to Stabilize transient protein complexes
In a landmark study, we developed Nbs that stabilize the β2AR-Gs complex. One of these Nbs that inhibits the GTP driven dissociation of β2AR-Gs was instrumental for obtaining the high-resolution crystal structure of this complex, providing the first view of transmembrane signaling by a GPCR. This structure was conducive in awarding the 2012 Nobel Prize in Chemistry to Robert Lefkowitz and Brian Kobilka. In two other studies, we developed Nbs that stabilize Vps34 complex II of yeast and the PINK1-Ubiquitin enzyme-substrate complex, respectively.
Unveiling conformational states of membrane pumps.
Functional understanding of membrane pumps requires the structural characterization of different conformational states, some with ligand-binding sites open to one side of the membrane and others with sites open to the other side. Nanobodies from our lab were instrumental to lock and solve the structures of inward-facing conformations of P-glycoprotein, LacY, an Fe2+ transporter, a fumarate transporter and the mitochondrial ADP-ATP transporter.
Nanobodies to investigate GPCR dynamics in vitro or inside cells.
Crystallographic studies provide static descriptions of highly dynamic behavior. In collaboration with several groups, we started applying our Nbs as versatile tools for the investigation of GPCR dynamics at the molecular and the sub-cellular level in vitro or as intrabodies inside cells.
Developing Nanobody-enabled conformational fragment screening.
By fusing Nanobodies that exhibit G protein-like behavior, we were able to lock β2AR, M2R or MOR constitutively in their active conformation. These GPCR-Nb fusions (ConFoFusions) have a unique pharmacological profile that is similar to the G protein bound receptor and allow the screening of fragments that selectively bind the active conformation of these receptors.
In 2015, the Steyaert lab founded Confo Therapeutics. Confo Therapeutics employs our proprietary CONFO® technology to lock inherently unstable functional conformations of GPCRs as a superior starting point for drug discovery. Confobody-stabilized active state conformations of these receptors reveal previously inaccessible structural features empowering the discovery of novel pathway-selective agonists for improved therapeutic intervention. Confo Therapeutics currently employs more than 50 people.
Developing Megabodies to expand cryo-EM analysis to proteins that are small and/or display preferential orientation in ice, two major factors that limit the resolution of reconstructed density maps. Megabodies are built by grafting nanobodies (~15 kDa) into larger scaffold proteins to produce stable, reasonably rigid and efficiently-folding monomeric chimeras. To proof this concept, such megabodies were used to solve the high resolution cryo-EM structure of the human synaptic GABAA receptor and uncover its signalling mechanisms.
Sharing technology with the European research community.
In 2014, we founded Nanobodies4Instruct as part of Instruct-ERIC, one of the first ESFRI projects that provides cutting-edge technology, scientific expertise and pioneering training. Any scientist from an Instruct member state can submit a proposal for access to the Nanobodies4Instruct center for the generation of conformational Nbs and now also megabodies to facilitate the structural analysis of proteins that are notoriously difficult to purify, crystallize or study by any other method.