Diamond blackfan anemia is a rare developmental disorder diagnosed by anemia, craniofacial anomalies, triphalangeal thumbs, bone marrow failure, and an increased cancer risk. It has been associated with mutation(s) in ribosomal proteins and/or biogenesis factor(s). Despite a clear association between mutations and DBA, there is no mechanistic description of how these mutations result in disease. Specifically, it is unclear how these mutations perturb the structural dynamics of uL18 and whether they are incorporated into the full ribosomal complex. To understand how uL18 structure is perturbed by DBA-associated mutations, we performed a total of 30μs molecular dynamics simulations of uL18 and nine DBA-associated variants in triplicates. Our simulations show that uL18 exists in two conformations: compact and extended, as determined by N-terminal domain (NTD) and C-terminal domain (CTD) docking into the core protein domain (CPD), stabilized by hydrophobic interactions. Our analysis suggests that DBA-associated variants impair the structural dynamics of uL18 in one of three ways: (1) destabilizing α-helix VI, (2) altering the conformational ensemble between the compact and extended conformations, and (3) altering the interaction interface of uL18 with ribosomal and non-ribosomal proteins. These findings suggest that these mutations lead to a destabilization of the protein by altering the conformational ensemble and perturbing the secondary structure. To validate our models, we are performing biophysical assays to study protein stability. To understand how uL18 haploinsufficiency impairs ribosomal structure, we performed cryo-EM studies. uL18 haploinsufficiency in Saccharomyces cerevisiae revealed ribosomes with missing density at the central protuberance, thus suggesting the presence of improperly assembled ribosomes. These insights will enhance our understanding of how point mutations in ribosomal protein uL18 lead to its haploinsufficiency, which in turn leads to structural abnormalities and is ultimately propagated to cause DBA pathogenicity.
Sapkota et al. (Sun,) studied this question.