Biomedical subjects

Roman C Hillig

Publications and source records attributed to Roman C Hillig.

6 recordsLinked to original sources

PubMed2006

Identifying protein construct variants with increased crystallization propensity--a case study.

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.

Cloning, Molecular↗

PubMed2004

A major histocompatibility complex-peptide-restricted antibody and t cell receptor molecules recognize their target by distinct binding modes: crystal structure of human leukocyte antigen (HLA)-A1-MAGE-A1 in complex with FAB-HYB3.

Antibodies with T cell receptor-like specificity possess a considerable diagnostic and therapeutic potential, but the structural basis of the interaction between an antibody and an histocompatibility antigen has so far not been determined. We present here the crystal structure (at 2.15 A resolution) of the recombinant, affinity-matured human antibody fragment Fab-Hyb3 bound to the tumor-associated human leukocyte antigen (HLA)/peptide complex HLA-A1.MAGE-A1. Fab-Hyb3 employs a diagonal docking mode resembling that of T cell receptors. However, other than these natural ligands, the antibody uses only four of its six complementarity-determining regions for direct interactions with the target. It recognizes the C-terminal half of the MAGE-A1 peptide, the HLA-A1 alpha1-helix, and N-terminal residues of the alpha2-helix, accompanied by a large tilting angle between the two types of molecules within the complex. Interestingly, only a single hydrogen bond between a peptide side chain and Fab-Hyb3 contributes to the interaction, but large buried surface areas with pronounced shape complementarity assure high affinity and specificity for MAGE-A1. The HLA-A1.MAGE-A1.antibody structure is discussed in comparison with those of natural ligands recognizing HLA.peptide complexes.

Amino Acid Sequence↗

PubMed2004

Design and synthesis of macrocyclic inhibitors of phosphatase cdc25B.

Based on molecular modeling studies, macrocyclic inhibitors of phosphatase cdc25B were synthetically derived from steroids. A preliminary SAR for this new template was elaborated. A series of compounds shows inhibition of cdc25B in the low micromolar range and good selectivity versus other phosphatases. The compounds did not show a significant antiproliferative effect in MaTu or HaCaT cells.

Cell Cycle Proteins↗

PubMed2003

Thermodynamic and structural analysis of peptide- and allele-dependent properties of two HLA-B27 subtypes exhibiting differential disease association.

Selected HLA-B27 subtypes are associated with spondyloarthropathies, but the underlying mechanism is not understood. To explain this association in molecular terms, a comparison of peptide-dependent dynamic and structural properties of the differentially disease-associated subtypes HLA-B*2705 and HLA-B*2709 was carried out. These molecules differ only by a single amino acid at the floor of the peptide binding groove. The thermostabilities of a series of HLA-B27 molecules complexed with nonameric and decameric peptides were determined and revealed substantial differences depending on the subtype as well as the residues at the termini of the peptides. In addition we present the crystal structure of the B*2709 subtype complexed with a decameric peptide. This structure provides an explanation for the preference of HLA-B27 for a peptide with an N-terminal arginine as secondary anchor and the lack of preference for tyrosine as peptide C terminus in B*2709. The data show that differences in thermodynamic properties between peptide-complexed HLA-B27 subtypes are correlated with a variety of structural properties.

Amino Acid Sequence↗

PubMed2002

HLA-B27 subtypes differentially associated with disease exhibit subtle structural alterations.

The reasons for the association of the human major histocompatibility complex protein HLA-B27 with spondyloarthropathies are unknown. To uncover the underlying molecular causes, we determined the crystal structures of the disease-associated B*2705 and the nonassociated B*2709 subtypes complexed with the same nonapeptide (GRFAAAIAK). Both differ in only one residue (Asp(116) and His(116), respectively) in the F-pocket that accommodates the peptide C terminus. Several different effects of the Asp(116) --> His replacement are observed. The bulkier His(116) induces a movement of peptide C-terminal pLys(9), allowing the formation of a novel salt bridge to Asp(77), whereas the salt bridge between pLys(9) and Asp(116) is converted into a hydrogen bond with His(116). His(116) but not Asp(116) adopts two alternative conformations, one of which leads to breakage of hydrogen bonds. Water molecules near residue 116 differ with regard to number, position, and contacts made. Furthermore, F-pocket atoms exhibit higher B-factors in B*2709 than in B*2705, indicating an increased flexibility of the entire region in the former subtype. These changes induce subtle peptide conformational alterations that may be responsible for the immunobiological differences between these HLA-B27 subtypes.

Aspartic Acid↗

PubMed2002

The complex of Arl2-GTP and PDE delta: from structure to function.

Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDE delta solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDE delta is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and G alpha(i1) interact with PDE delta and that, at least for H-Ras, the intact C-terminus is required. We suggest PDE delta to be a specific soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDE delta-mediated transport.

Amino Acid Sequence↗