COPZ1 mutations in 3 patients with CN. (A-B) Family trees and corresponding Sanger sequencing traces showing the inheritance of the homozygous stop-codon mutation c.445C>T:p.Gln141Ter (A) or MS mutation c.394G>C, p.Gly132Arg. (B) in COPZ1 in patients (P1-P3) with CN. Red arrows indicate positions of mutations. (C-D) Bone marrow smears of patients P1 (×100 original magnification) and P3 (×50 original magnification). (E) Neighbor-joining phylogenetic tree of COPZ1 proteins, performed with 1000 bootstrap replications. (F) Schematic representation of the COPZ1 gene (top) and COPZ1 protein (bottom) sequences highlighting protein domains, and positions of mutations. (G) Representative western blot images of COPZ1 protein in lysates of WT or COPZ1-TR K562 cells. α-tubulin staining was used to control protein loading. (H-I) RMSF plots of free full-length COPZ1 (H) and COPZ1-TR (I) show structural destabilization of the C-terminal segmented COPZ1 upon truncation. Purple dots and green shades represent the Cɑ RMSF averages and standard deviations from 3 independent simulations. (J) Estimation of backbone entropy change across the 4 states of free, full-length COPZ1 (upper left), free COPZ1-TR (upper right), bound COPZ1 full length (lower left), and bound COPZ1-TR (lower right) indicate a higher entropic penalty on the COPZ1-TR upon binding to COPG1. Large putty cartoons show the average Cɑ RMSF in tube width and color (in ascending order, cyan, blue, magenta, and red). Small cartoons show the whole model, in which COPZ1 is cyan, COPG1 is yellow, and the last backbone atom in COPZ1 is shown as a purple dot. (K) Quantifying the COPZ1:COPG1 interaction energies shows no difference in COPG1 binding by either COPZ1 or COPZ2. Moreover, an apparent loss of interaction energy is observed for COPZ1-TR, which is on par with previously described interaction mutants (COPZ1 mut1 and mut4).25 (K-L) The COPZ1-MS mutation, however did not cause any difference in either binding interactions with COPG1 (K) or structural stability in the unbound form (L). Ca, Carassius auratus; Ce, Caenorhabditis elegans; Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus; Ol, Oryzias latipes; Pt, Pan troglodytes; RMSF, root mean square fluctuation.

COPZ1 mutations in 3 patients with CN. (A-B) Family trees and corresponding Sanger sequencing traces showing the inheritance of the homozygous stop-codon mutation c.445C>T:p.Gln141Ter (A) or MS mutation c.394G>C, p.Gly132Arg. (B) in COPZ1 in patients (P1-P3) with CN. Red arrows indicate positions of mutations. (C-D) Bone marrow smears of patients P1 (×100 original magnification) and P3 (×50 original magnification). (E) Neighbor-joining phylogenetic tree of COPZ1 proteins, performed with 1000 bootstrap replications. (F) Schematic representation of the COPZ1 gene (top) and COPZ1 protein (bottom) sequences highlighting protein domains, and positions of mutations. (G) Representative western blot images of COPZ1 protein in lysates of WT or COPZ1-TR K562 cells. α-tubulin staining was used to control protein loading. (H-I) RMSF plots of free full-length COPZ1 (H) and COPZ1-TR (I) show structural destabilization of the C-terminal segmented COPZ1 upon truncation. Purple dots and green shades represent the Cɑ RMSF averages and standard deviations from 3 independent simulations. (J) Estimation of backbone entropy change across the 4 states of free, full-length COPZ1 (upper left), free COPZ1-TR (upper right), bound COPZ1 full length (lower left), and bound COPZ1-TR (lower right) indicate a higher entropic penalty on the COPZ1-TR upon binding to COPG1. Large putty cartoons show the average Cɑ RMSF in tube width and color (in ascending order, cyan, blue, magenta, and red). Small cartoons show the whole model, in which COPZ1 is cyan, COPG1 is yellow, and the last backbone atom in COPZ1 is shown as a purple dot. (K) Quantifying the COPZ1:COPG1 interaction energies shows no difference in COPG1 binding by either COPZ1 or COPZ2. Moreover, an apparent loss of interaction energy is observed for COPZ1-TR, which is on par with previously described interaction mutants (COPZ1 mut1 and mut4).25 (K-L) The COPZ1-MS mutation, however did not cause any difference in either binding interactions with COPG1 (K) or structural stability in the unbound form (L). Ca, Carassius auratus; Ce, Caenorhabditis elegans; Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus; Ol, Oryzias latipes; Pt, Pan troglodytes; RMSF, root mean square fluctuation.

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