The V119I polymorphism in protein L-isoaspartate O-methyltransferase alters the substrate-binding interface

Protein L-isoaspartate O-methyltransferase (PIMT)repairs isoaspartate residues in damaged proteins, and itcontains a Val–Ile polymorphismin in a5, 13 A˚ from itsactive site. Val119 has lower activity and thermal stabilitybut increased afﬁnity for endogenous substrates. Studiessuggest that heterozygosity for Val/Ile favors efﬁcient iso-aspartate repair. We have performed multiple moleculardynamics simulations of 119I and 119V PIMT. Both V119and I119 interact with the same residues throughout allof the simulations. However, the larger Ile altered theorientations of a5andb5, both of which have co-substratebinding residues on their distal ends. I119 increases theﬂexibility of several residues, loosening up the S-adenosyl-methionine (SAM)-binding site. These subtle changes arepropagated towards the isoaspartate-docking site via resi-dues common to both active sites. The increased mobilityin 119I PIMT reorients a3, resulting in a salt-bridgenetwork at the substrate-binding interface that disruptsseveral key side-chain interactions in the isoaspartate site.In contrast, 119V PIMT remains quite rigid with littlechange to the co-substrate binding site, which could hinderSAM’s binding and release, accounting for the decreasedactivity. These results shed light on the molecular basisbehind the decreased activity and increased speciﬁcity forendogenous substrates of 119V PIMT relative to the 119Ivariant. 119I PIMT catalyzes the methylation reaction butmay have difﬁculties recognizing and orienting speciﬁcsubstrates due to its distorted substrate-binding site.Heterozygosity for both the Ile and Val alleles mayprovide the best of both worlds, allowing the fast andspeciﬁc methylation of damaged proteins.Keywords: aging/molecular dynamics/polymorphisms/protein isoaspartate O-methyltransferase/protein repairIntroductionAsparagine residues undergo spontaneous deamidation toform L-isoaspartate, kinking the neighboring main chainstructure through the addition of an extra methylene group inthe polypeptide backbone (Najbauer et al., 1996; Aswadet al., 2000; Reissner and Aswad, 2003; Shimizu et al.,2005). This process is exacerbated by heat shock and oxi-dative stress, and it is a major source of protein damage. Theaccumulation of isoaspartate residues has been shown toaffect the function of several cellular proteins, includinghistone H2B (Young et al., 2001, 2005), Bcl-x (Devermanet al., 2002), protein kinase A (Pepperkok et al., 2000), col-lagen type 1 (Lanthier and Desrosiers, 2004) and ribosomalprotein S11 (David et al., 1999). Isoaspartate residues havealso been found in long-lived proteins such as eye-lensb-crystallin (Voorter et al., 1988; Fujii ., 1994) andmyelin basic protein (Fisher et al., 1986; Shapira .,1988). The buildup of isoaspartate residues in amyloid Abpeptide and erythrocytic membrane-proteins, includingankyrin, have been associated with Alzheimer’s Disease(Shimizu et al., 2000, 2005) and Down’s syndrome (Gallettiet al., 2007), respectively.Protein L-isoaspartate O-methyltransferase (PIMT,EC.2.1.1.7) is a ubiquitous housekeeping protein that cata-lyzes the ﬁrst step of isoaspartate repair by transferring amethyl group from co-substrate S-adenosylmethionine(SAM) to the a-carboxyl group of the L-isoaspartate residue(Clarke, 2003; Reissner and Aswad, 2003; Shimizu et al.,2005). PIMT is highly expressed in developing neural tubes,the hippocampus and cerebral cortex, and it is essential formaintaining proper CNS function (Diliberto and Axelrod,1976; Mizobuchi et al., 1994; Shirasawa et al., 1995;Lanthier et al., 2002). PIMT knockout mice accumulate 4–8-fold more isoaspartate-containing proteins in the brain rela-tive to wild-type mice, leading to atypical neurotransmission,developmental defects and fatal seizures within 2 months ofbirth (Kim et al., 1997; Zhu et al., 2006b). In addition, over-expression of PIMT in drosophila melanogaster has beenshown to increase the organism’s lifespan by 32–39%(Chavous et al., 2001).PIMT has a common valine ! isoleucine polymorphismat residue 119 (Ingrosso et al., 1989; Tsai and Clarke, 1994).The 119V and 119I alleles occur with approximately equalfrequencies in Caucasian populations, whereas 119I (theancestral allele) is dominant in Asian and African popu-lations (Tsai and Clarke, 1994; DeVry and Clarke, 1999).The speciﬁc activity of 119I PIMT is 20% higher than thatof the 119V protein in red blood cells (David et al., 1997;DeVry and Clarke, 1999). The activity of 119I PIMT is alsomore thermo-tolerant and less sensitive to oxidative stressthan that of 119V PIMT (David et al., 1997). Although thereappears to be no difference in the afﬁnities of 119I and 119VPIMT for exogenous peptides (Lowenson and Clarke, 1991),the 119V protein has an 30% higher afﬁnity for endogen-ous substrates found in the brain cytosol of PIMT knockoutmice (DeVry and Clarke, 1999). In addition, Lowenson andClarke (1991) showed that despite the higher speciﬁc activityof 119I PIMT, the accumulation rate of damaged proteins isgoverned by substrate afﬁnity. These data suggest that com-bining the 119I protein’s increased activity and stability withthe 119V variant’s improved substrate afﬁnity may result inthe most effective repair of isoaspartate-mediated protein