Journal:Proteins:2

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.

. Domains are: regulatory (yellow); catalytic (green); tetramerization (blue). The substrate L-Phe (substrate analog beta(2-thienyl) alanine (TIH) is shown) and cofactor tetrahydrobiopterin (BH4 or H4B) both have binding sites in the catalytic domain. The TIH, Fe (++) ion and cofactor Tetrahydrobiopterin (BH4) are shown space filled.

Category 1: 28 missense mutations are expected to affect stability only

28 of the 35 mutations with destabilization assignments are remote from any known ligand binding or the catalytic site, and so are expected to have a low experimental protein level, and wild type specific activity.

, in orangered) have protein levels less than 50% wild type, as expected. Of these, all but two have wild type specific activity. The two exceptions, , have approximately three fold higher specific activities than the wild type. These mutations lie in the regulatory domain, suggesting a possible explanation for the high activity level. The 16 mutants are classified into clinical categories of mild PKU (A104D), moderate PKU , and classic PKU .

Selected mutations from this group:

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Mutations R252G/Q/W caused saltbridge lost and hydrogen bonds lost:

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Nine of remaining mutations expected to affect stability only , in blueviolet) have reported experimental protein levels greater than 50% of wild type (all 100%, except one of the R408Q experiments with 70%), inconsistent with the computational assignment.

Selected mutations from this group:

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Category 2: Seven missense mutations are expected to affect both stability and molecular function

There are seven mutations , in magenta) with atomic contacts of 6.5 Å or less to the , and that are assigned as destabilizing by the structure SVM. Residues interacting with TIH, BH4 and the Fe++ ion are in green. Water molecules shown as red spheres. These mutant proteins are therefore expected to exhibit a combination of lower specific activity and a lower total protein level. Six of the seven (G247V, L255S, R270S, E280K, S349L, S349P) have protein levels less than half or in one case close to half (G247V, 56%) that of wild type, and very low protein activity, consistent with expectations. Clinical categories are available for E280K, S349L, and S349P, and are all “classic PKU”, consistent with the results and with experiment. The remaining mutant in this category, Y277D, has an experimental activity of zero, and is classified as mild or classic PKU, consistent with the profile SVM assignments. But the measured protein level is reported as 99% of wild type, inconsistent with a modest confidence stability assignment. This may be a computational false positive with respect to stability.

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Category 3: Nine mutations are expected to impact molecular function only

A total of nine mutations are classified as high impact by the sequence conservation method, classified as not destabilizing by the stability method, and so are expected to impact molecular function but not stability, implying wild type protein levels and lower activity. Four of these, (in cyan) have atomic contacts of 6.5 Å or less to a ligand. Experimental data for two, A322G and L348V, are consistent with expectations, with low activity and normal protein levels. The remaining two, L255V and P281L, have low activity, but also low protein level. Both are in direct contact with the BH4 cofactor, and would disrupt binding substantially:

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The other five mutations in this category, (in deepskyblue), are not near to any known ligand binding or catalytic site.

Selected mutations from this group:

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Category 4: Two mutations are assigned low impact by both the sequence conservation and stability methods

Two mutations, (in darkmagenta), are assigned low impact by both computational methods. Both sets of experimental results show close to normal activity and protein levels, consistent with the analysis results. Also reasonably consistent, T92I is assigned to the mild MHP category of disease, suggesting a subtle effect on protein function. Inconsistent with both experiment and computational analysis, P211T is assigned to the “classic PKU” category, based on a single functionally hemizygous patient genotype.

Evolutionary conservation