Combination of multiple alignment analysis and surface mapping paves a way for a detailed pathway reconstruction—The case of VHL (von Hippel‐Lindau) protein and angiogenesis regulatory pathway

Using the tumor suppressor VHL protein as an example, we show that detailed analysis of conservation versus variation pattern in the multiple alignment can be coupled with the genomic pathway/complex conservation analysis to provide a more complete picture of the entire interaction/regulatory network. Results from the present study have allowed us to hypothesize that two additional proteins are involved in the VHL‐mediated regulation of angiogenesis. Detailed modeling also has led to a prediction of the possible interaction mode between the known and the proposed parts of the VHL complex. To aid in an analysis of the VHL protein regulation of HIF‐1α degradation, an important and only partially understood process that directly influences angiogenesis, we performed a comprehensive search for the orthologs of the VHL as well as for VHL‐interacting proteins in all the available eukaryotic genomes. Analysis of a multiple alignment of thus identified VHL orthologs reveals an unusually high degree of conservation of the surface amino acid residues that almost exactly correspond to positions mutated in the VHL disease‐associated tumors. In addition, these positions form well‐defined clusters in three‐dimensional space, and presence or absence of individual clusters correlates with the presence or absence of pathway elements in different genomes. We have also shown that relation trees derived from the multiple sequence alignment, functional surface‐mapping, and HIF‐1α degradation pathway structure are in complete agreement, linking the functional and structural evolution of the VHL protein and VHL‐dependent HIF‐1α degradation complex.