Predicting Structural and Serologic Modifications in Blood Group Antigens by Homology Modeling.

Homology modeling of blood group proteins offers the possibility of predicting the effect of amino acid changes on serologic phenotype and immunogenicity. The location of an amino acid change within known structural motifs, its phylogenetic conservation, and its proximity to known epitopes give insight into its potential effect on protein structure and, consequently, its clinical significance. We applied this approach to investigate the loss of membrane expression of the Dombrock blood group antigens in a patient with a single amino acid change and to investigate RhD alterations in weak D phenotypes. The Dombrock homology model was derived with rat ART2.2 crystal structure as template. For the RhD model, the crystal structure of the Rh-like-ammonia transporter from Nitrosomonas europaea was used. Protein alignment was derived with Clustal X, adjusted visually, and submitted to the Swiss Modeling server. Models were viewed with Deep View Swiss Pdb Viewer. The Dombrock null containes a Phe62Ser substitution. This Phe (F) residue is located in an FDDQY motif near the COOH terminus. This region of the protein also contains a HYYLT motif. These two motifs are highly conserved in the ART protein family and contribute several aromatic amino acids to this region of the molecule. Aromatic side chain interactions between these residues could contribute to the stability of the Do protein. In support, the distance in the ART2.2 crystal structure between Phe in FDDQY and His in HYYLT is 3.7 Å, which is the appropriate distance for aromatic side chain interactions. This is also the measured distance between these two residues in the Do model. Thus, protein modeling indicates that the Phe62Ser mutation disrupts important stacking interactions between Phe62 and His160. When amino acid changes causing weak D phenotypes were examined, some of those affecting expression of RhD were located near the vestigial transport channel. These include the Trp220Arg mutation (weak D Type 16). This Trp residue is part of the transport channel in Nitrosomonas and is conserved in Rh proteins of almost all species. Its role in maintaining Rh structure is indicated by the dramatic effect its modification has on protein and epitope expression. Additionally, Arg114Trp change (weak D Type 17), which is also near the channel, reduces D expression to only 66 antigen sites/cell. GlyXXXGly motifs stabilize interactions of adjacent alpha helices in membrane proteins. Evidence for a role in stabilization of RhD is revealed by the Gly282Asp mutation (weak D Type 15) which is part of such a motif. In addition, a D-epitope in loop 3 is near the 282Asp residue. Alteration of helical packing accompanied by epitope conformation could explain production of anti-D in patients with weak D Type 15. Homology modeling is an important tool for understanding the structure and serologic bases of blood group proteins and will continue to give important insight as more protein crystal structures become available.