Crystallization chaperones

We generate nanobodies that facilitate the crystallization of challenging targets and identifies nanobodies that stabilize unique conformations to study the structure, function and dynamics of these proteins.

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Structural enzymology

We are interested in understanding how enzymes function at the molecular and atomic level. We combine structural biology with protein engineering and enzyme kinetics to study how structure determines function. 

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Protein-protein interactions

Protein-protein complexes play an important role in biology and disease. In order to characterise these "transient" protein-protein interactions, we are developping Nanobody-based methods to stabilize PPIs.
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Structural enzymology

Wim Versees
Project Leader

 

We are interested in understanding how enzymes function at the molecular and atomic level. We combine structural biology with protein engineering and enzyme kinetics to study how structure determines function. Also here, Nanobodies are used throughout as versatile tools to stabilize and crystallize flexible enzymes or subunits from large enzyme complexes.

Current research topics include analysis of the structure and function of bacterial GTPases and of enzyme complexes involved in tRNA modification.

 

 

tRNA modification

 

Post-transcriptional tRNA modifications play a primordial role in the translation process as they influence tRNA stability and folding, cognate codon recognition, stabilization of the codon-anticodon wobble base pairing and correct aminoacylation. In the last years, the awareness is growing that post-transcriptional tRNA modifications, especially at the wobble position, might regulate important cellular processes at the level of protein translation. Concomitantly a number of tRNA modification reactions are catalyzed by large enzyme complexes that are regulated via G protein modules or through phosphorylation. On of our goals is to decipher the structure, function and regulation of these enzyme complexes in order to contribute to our understanding of their cellular roles and the way they are incorporated in signaling networks.

 

 

  • The Elongator complex

 

This eukaryotic six protein complex was initially identified as an Elongation factor in transcription. However, in the last years new cellular functions for this enzyme complex are been identified at an amazingly high pace (Versées et al., 2010). It is well established that the genes coding for the Elongator complex are all required for an early step in the biosynthesis of the 5-methoxycarbonylmethyl-uridine (mcm5U) or 5-carbamoylmethyl-uridine (ncm5U) wobble modification of 11 different tRNA species. However, the contribution of the individual proteins is unknown and the molecular mechanism of the tRNA modification reaction has not yet been established.

Recently, the group of P. Verstreken showed in collaboration with our group and the group of W. Robberecht that yet another function of this complex involves the acetylation of the protein Bruchpilot at nerve cell active zones. This function might link to the involvement of Elongator in the neurological disease ALS (Miśkiewicz et al., 2011).

 

  • tRNA methyltransferases

 

Methyltransferases form a major class of tRNA modifying enzymes needed for the proper functioning  and the stability of tRNA. Known RNA MTases can be classified into four superfamilies, including Rossmann-fold (RFM), SPOUT (SpoU and TrmH), Radical-SAM and FAD/NAD(p)-dependent methyltransferases. While some methyltransferases catalyze the methyl transfer reaction using a catalytic domain alone, others are fused to one of the various RNA binding domains. In collaboration with the group of L. Droogmans (ULB, Belgium) we are investigating the structure and function of a variety of methyltransferases in order to get a better understanding of their tRNA specificity and the contribution of their catalytic and RNA-binding domains to catalysis and substrate binding.

 

 

Bacterial GTPases

 

Guanine nucleotide binding proteins (GNBPs or G proteins) are involved in many essential cellular processes such as protein synthesis and translocation, membrane trafficking, signal transduction and cell cycle control. While GNBPs are ubiquitously found in eukaryotes, a core of only 13 classes of G proteins have been found to be universally conserved among bacteria and nearly all of them seem to elicit their function through interaction with RNA and/or the ribosomes (Verstraeten et al., 2011). Eukaryotic GTPases have been well studied, and their activity is often regulated through the action of auxiliary proteins (Guanine nucleotide Exchange Factors and GTPase Activating Proteins). In contrast, very little is known on the function, molecular mechanism, regulation and effectors of bacterial GTPases.

 

  • The MnmE-GidA complex

 

The MnmE/GidA complex is a low affinity protein complex involved in tRNA wobble modification that is conserved from bacteria to man. MnmE is one of the highly conserved G proteins, belonging to a newly described class of G proteins activated by (homo)dimerisation (Gasper et al., 2009). Mutations in the human ortholgs (termed GTPBP3 and Mto1) have been implicated in respiratory defects leading to severe mitochondrial myopathies and non-syndromic deafness. In several bacteria, MnmE and GidA have been shown to regulate the expression of virulence factors on a translational level.

The crystal structures of MnmE and GidA outside the complex have been solved and biochemical data has shown that MnmE and GidA form a relatively low affinity complex where large conformational changes throughout the complex, induced by the nucleotide state of MnmE, are required for the tRNA modification (Meyer et al., 2009). Despite this biochemical and structural information on the individual proteins, the fine details of the tRNA modification reaction, the structure of the MnmE/GidA complex and the nature and relevance of the GTPase-driven conformational changes remain thus far enigmatic. For the elucidation of the structure and function of the MnmE-GidA complex we collaborate with the group of A. Wittinghofer (MPI Dortmund).