Research Group Yves Muller


Protein structure and protein design

De novo structure determination using X-ray crystallography

Structure-function-relationships in plasma transport proteins
Corticosteroid-binding globulin (CBG) transports cortisol, corticosterone and progesterone in the blood and regulates the tissue availability of these hormones. Humans exhibiting CBG variants show symptoms of hypotension and chronic pain. We have contributed the 3D structure of rat CBG revealing a fold very similar to serine protease inhibitors (SERPINs). Just like SERPINs, CBG contains a reactive center loop (RCL) which is targeted by proteases. RCL cleavage leads most likely to structural rearrangements and reduced steroid-binding affinity. Besides X-ray crystallography, circular dichroism spectroscopy and isothermal titration calorimetry are used to study structure-function-relationships in CBG. A further crystal structure of human CBG in complex with progesterone could explain the lower affinity of progesterone in comparison to corticosteroids. Additional structures, such as the crystal structures of uncleaved rat and human CBG with no steroid bound, are necessary to fully decipher the molecular mechanisms underlying CBG function.

Human apolipoprotein M (ApoM) is located mainly in the plasma high density lipoprotein (HDL) fraction. Studies in apoM gene-modified mice indicated ApoM to be antiatherogenic, however, its physiological role is understood only fragmentary. As a ligand, sphingosine-1-phosphate (S1P) is provided by HDL-associated ApoM. The crystal structure of human ApoM in complex with S1P revealed the determinants of S1P-binding specificity. Furthermore, we could show on the basis of structural and functional studies, how ApoM combines ligand dissociation with HDL anchoring (see the following model for the association of ApoM with HDLs). Future work aims at solving a ligand-free ApoM structure providing a framework for computational ligand docking experiments and possibly drug design.


Host/pathogen interactions: Structural biology of Salmonella and cytomegalovirus proteins
Salmonella infections are caused by ingestion of contaminated food and water. SiiE, a giant 595 kDa non-fimbrial adhesin from Salmonella enterica initiates the adhesion to polarized host cells. Bioinformatics analyses predict SiiE to contain 53 bacterial Ig-like (BIg) domains, as well as a region containing heptad-repeats at the N-terminus and two ca. 50-residue-long insertions flanking the C-terminal BIg domain 53. The BIg domain architecture could be verified by solving the 3D structure of a fragment comprising BIg domains 50, 51 and 52. Furthermore, this structure revealed two types of SiiE-specific calcium-binding sites, either present in the domain interfaces or comprised within single domains. Molecular dynamics simulations and electron microscopy images in presence and absence of Ca2+ suggest that Ca2+ rigidifies consecutive pairs of BIg domains leading to a rigid rod-like overall structure.

Immediate early protein 1 (IE1) is one of the earliest viral proteins expressed in host cells upon lytic cytomegalovirus (CMV) infection. Nuclear domains 10 (ND10) are large nuclear protein complexes that participate in intrinsic cellular defense mechanisms against pathogens. IE1 is able to disrupt and dissolve the ND10 protein complexes and this possibly represents a key mechanism by which herpesviruses attempt to overcome the host cell defense mechanism. We currently work on the elucidation of crystal structures of either IE1 by itself or in complex with host proteins.

Insight into the allosteric regulation of bacterial repressors
The aim of this project is to elucidate structure-function relationships in various transcriptional regulators, among them YvoA from Bacillus subtilis. YvoA is mainly implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its crystal structure provides detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription. Central to this allosteric coupling mechanism is a loop-to-helix transition, which leads finally to a 122° rotation of the DNA-binding domains that is best described as a jumping-jack-like motion (see the following figure). Crystal structures of YvoA in complex with glucosamine-6-phosphate and N-acetylglucosamine-6-phosphate provide for the first time detailed atomic insight into the specific recognition and coordination of bound effector molecules. In addition, we have solved the 3D structure of isolated YvoA-DNA-binding domains in complex with palindromic dsDNA, revealing both the determinants for highly sequence-specific operator dre-site (specific DNA operator sites) recognition and for the unspecific binding of YvoA to dsDNA.


Probing protein architecture / protein design

Current advances in computational protein design enable the de novo design of proteins with novel properties. We have implemented established protein packing algorithms in the computer program MUMBO and are currently investigating the validity of these approaches by transforming a homodimeric bacterial repressor into a chain-specific heterodimer, redesigning the specificity of enzymes (for example SDRs) and transferring ligand-binding sites between different protein scaffolds (for example between plasma transport proteins). The program MUMBO is freely available to academic users upon request.