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The 3-D models also allow to visualise the structural consequences that the crd mutant alleles impart to the PsYUC1 protein. The crd-1 mutant has leaf vasculature phenotypes (McAdam et al., 2017b; Swiecicki, 1989). However, no change in overall structure and folding pattern are predicted at the tertiary protein level. The

crd-1 point mutation, located at the first ATG motif (McAdam et al., 2017b),

changes a glycine to an aspartic acid residue (G142D) (Figure 3.12). Glycine does not have any side chain, increasing the flexibility of the polypeptide chain at that location. Glycine, being neither hydrophilic nor hydrophobic, can be found in either environment but is generally considered non-polar (McMurry and Simanek, 2007). Residues in the vicinity of Gly 142 are mainly hydrophobic (Val 138 and 139, Ala 140 and Ala 145). However, the amino acids directly flanking Gly142 are polar (Thr 141 and Asn 144) and negatively charged (Glu 143) implying that Gly 142 may assist in increasing contact with water.

The change of glycine to aspartic acid (G142D) does not appear to affect the PsYUC1 folding pattern or torsion angle at the location of the amino acid change (Figure 3.12). Aspartic acid, the amino acid coded for by crd-1, has a negatively charged side chain. Potentially, having two negatively charged amino acids side-by- side may increase polarity within that specific area, allowing too much water to be incorporated thus preventing Thr 141 and Asn 144 from forming efficient hydrogen bonds with the ligand. Furthermore, at position 142, all other 19 amino acids have a SIFT score (sift.jcvi.org/sift) of 0.00 (<0.05 considered deleterious), with the exception of glycine being the only amino acid present and tolerated in the 48 sequences included in the alignment. Any changes to glycine at position 142 is expected to have aberrant consequences on the functionality of the protein.

Figure 3.12. PsYUC1 and crd-1 predicted protein structure. a and b) PsYUC1 structure with close-up of location of G142. c and d) The location of the altered amino acid D142. Generated with Swiss-Model server.

The crd-2 mutation, originally referred as crd-whip (Berdnikov et al., 2000), has a

single nucleotide substitution (G628A) resulting in a frameshift at position 210 in the translated protein sequence. Two principal protein products, with altered sequences and premature termination are produced due to alternative splicing of the mutant RNA: the longer product terminates at position 262 and the shorter product, the most abundant of the two, terminates at position 244 (McAdam et al., 2017b). The products have several amino acids predicted as deleterious. The shorter product has five amino acid out of 35 with SIFT scores below 0.05 and the longer product, has eight out of 53. Both frameshifts and early truncations adversely affect the proteins

folding patterns and structures. The secondary structures of the most abundant product, the shorter crd-2, and the product used in all proceeding predictions, is disrupted from -helix H onwards (Figure 3.13). The second ATG motif (Hou et al., 2011) is missing as are the last five -helices and seven -strands.

Figure 3.13. Comparison of the predicted secondary structures of the C-terminal section of the PsYUC1 and crd-2. The sequence section affected by the crd-2 is shown with grey triangles. Confidence levels are colour coded. Secondary structures generated with the Phyre2 web portal (sbg.bio.ic.ac.uk/~phyre/).

The C-terminal section of the crd-2 protein sequence is missing, which consequently translates to the 3-D in silico structure having fewer FAD and NADPH binding residues being predicted (Figure 3.14). Many of the FAD-related binding amino acids can be seen in the pocket as these principally occur in the ‘Eastern’ side of the protein (Figure 3.11b, c). However, in space-filling mode, much of the FAD cofactor and its associated binding residues are not fully incorporated in the crd-2 pocket when compared with the WT PsYUC1 structure (Figure 3.11d). The PsYUC1 protein from the crd-3 mutant (line FN1522-1) cannot be modelled due to

the complete deletion of PsYUC1 from the genome. Consequently, crd-3 can be confidently presumed to be a null mutant (McAdam et al., 2017b).

Figure 3.14. The protein structure of the crd-2 mutant and its predicted binding amino acids for the FAD and NADPH cofactors. a) Viewed from above, the WT protein is presented in ribbon mode. The predicted binding residues are in dark blue and areas that are affected in crd-2 are encircled. b) The crd-2 protein presented in ribbon. Areas where secondary structures are missing are encircled. c and d) The structure (grey), the binding residues (blue) and the FAD and NADPH ligands (green) in space-filling mode for PsYUC1 and crd-2 respectively. d) FAD and associated binding residues are not quenched in the pocket. Generated with Phyre2.

For crd-4, a single-point mutation (G1730A) results in a frameshift at position 334 of

the protein sequence (McAdam et al., 2017b). The resulting product has a premature termination at position 361 and 27 altered amino acids, 12 of which are predicted as deleterious with SIFT scores < 0.05. Despite the fact that the crd-4 mutation occurs

downstream of the consensus FMO domains, the protein structure is nonetheless adversely affected (Figure 3.15). The last two -helices and 17 are missing.

Figure 3.15. Comparison of the predicted C-terminal secondary structures of PsYUC1 and crd-4. The sequence section affected by the crd-4 is shown with grey triangles. Confidence levels are colour coded. Secondary structures generated with the Phyre2 web portal (sbg.bio.ic.ac.uk/~phyre/).

The physical changes caused by the crd-4 allele alter the secondary structures at the C-terminal end of the PsYUC1 protein (Figure 3.16a, b). Folding patterns are not substantially modified but the missing -strand and -helices may affect the capacity of the binding pocket to interact efficiently with the ligands. Indeed, in space-filling mode, much of the FAD cofactor and its associated binding residues are not fully incorporated in the crd-4 pocket (Figure 3.16c, d).

Figure 3.16. The effects of the crd-4 mutation on the PsYUC1 architecture. a and b) Tilted 30 backwards, the PsYUC1 and crd-4 proteins are presented in ribbon mode. The predicted binding residues (dark blue) and FAD and NADPH ligands (green) are shown as stick models. The region affected in the crd-4 mutant is encircled. c and d) The whole structures are viewed from the front in space-filling mode. Generated with Phyre2.

In the WT PsYUC1 protein, Loop-30, found between -strand 17 and -helix N, is located above the floor of the binding pocket, -helix B, and folds back onto the area where the FAD cofactor locates (Figure 3.17a). The ‘folding back’ of Loop-30 appears to form a ‘roof’ or a ‘lid’ above the binding pocket. The hydrophobic amino acids associated with Loop-30 (Val 361, Phe 363 and Leu 368) face down towards the pocket (Figure 3.17b). Val 361 is proximally placed above Gly 20, a canonical FAD binding residue of FMOs (Eswaramoorthy et al., 2006). The two arginine (Arg 365 and 366) side chains outstretch over the binding site. Arginine is amphipathic, meaning that the positively charged head of the side chain possibly interacts with the

environment outside of the protein while the hydrophobic hydrocarbon chain may interact with the interior and the FAD cofactor.

Figure 3.17. Loop-30 may be important to the functionality of the PsYUC1 protein. a) Loop-30 is situated above the binding pocket. Lys 333 is selected because Gly 334, the start of the crd-4 frameshift, is located behind Lys 333 and cannot be seen in this orientation. b) Amino acids that form Loop-30. Oxygen in red, nitrogen in blue and hydrogen in light grey. Generated with Swiss-Model.

b a

Loop-30, and the amino acids flaking each side, are highly conserved in plants but not in bacterium or in animals, represented here by a rabbit FMO (Figure 3.18). Potentially, in plant species, Loop-30 and its flanking residues, behave in a similar fashion as another protein with a ‘lid’. Indeed, a simpler protein, Hsp70 or DnaK, a chaperone involved in protein export destined for secretion in E. coli (Hartl, 1996; Wild et al., 1992), has a lid located above the binding pocket. The lid did not appear to have binding properties per se, but was demonstrated to ‘capture’ the substrate within the binding domain (Zhu et al., 1996). Further studies demonstrated that the removal of the lid drastically reduced the binding capacity of the protein by

destabilising the pocket and reducing its affinity for the substrate (Moro et al., 2004).

Figure 3.18. Sequenced alignment of the C-terminal end of YUC1-related proteins from a range of species, including eudicots, monocots, bacterium and mammal. The second ATG motif (LATGY), the location of the crd-4 mutation (red arrow) and several tertiary structures are included as references. The pink box marks the amino acids that form the binding pocket ‘lid’ and include β-strand 17, Loop-30 and part of α-helix N. The two Arginine, in red, are located at the centre. Colouring scheme represents the amino acid family. Aligned with ClustalW and presented with the EMBL-EBI MVIEW website.

3.3.6. The crd-4 mutant also carries the Pstar1-1 mutation