Protein Stability and in Vivo Concentration of Missense Mutations in Phenylalanine Hydroxylase
Zhen Shi, Jenn Sellers, and John Moult [1]
Molecular Tour
A previous computational analysis of missense mutations linked to monogenic disease found a high proportion of missense mutations affect protein stability, rather than other aspects of protein structure and function. The purpose of the present study is to relate the presence of such stability damaging missense mutations to the levels of a particular protein present under in vivo like conditions, and to test the reliability of the computational methods. Experimental data on a set of missense mutations of the enzyme phenylalanine hydroxylase (PAH) associated with the monogenic disease phenylketonuria (PKU) have been compared with the expected in vivo impact on protein function, obtained using SNPs3D, an in silico analysis package. A high proportion of the PAH mutations are predicted to be destabilizing. The overall agreement between predicted stability impact and experimental evidence for lower protein levels is in accordance with the estimated error rates of the methods. For these mutations, destabilization of protein three dimensional structure is the major molecular mechanism leading to PKU, and results in a substantial reduction of in vivo PAH protein concentration. Although of limited scale, the results support the view that destabilization is the most common mechanism by which missense mutations cause monogenic disease. In turn, this conclusion suggests the general therapeutic strategy of developing drugs targeted at restoring wild type stability.
Under physiological conditions, , with each subunit composed of three domains. From N terminal to C terminal these are the regulatory, catalytic and tetramerization domains. To date, no experimentally determined structure of the complete human molecule is available. Three PDB structures were selected to model specific mutations in different domains based on crystal structure resolution, structure quality, and coverage: 1j8u, (monomeric form); 2pah, (tetrameric complex); and 1phz, (dimeric complex). The high resolution human 1j8u structure was used to model catalytic domain mutations. Regulatory domain mutations were modeled using a homology model of the human domain, based on the rat 1phz structure, as were three catalytic domain mutations, R261Q, R413P, and Y414C, that are in contact with the regulatory domain across a subunit interface. Rat PAH protein has 93% sequence identity with human PAH. There are no insertions or deletions in sequence between the two proteins. Main chain coordinates were taken directly from the rat structure. Side chains conformations were optimized using SCRWL. Catalytic domain mutations R408W and R408Q are in contact with the tetramerization domain of another subunit and were modeled using 2pah.
