Sequence determinants of thermodynamic stability in a WW domain—An all‐β‐sheet protein

The stabilities of 66 sequence variants of the human Pin1 WW domain have been determined by equilibrium thermal denaturation experiments. All 34 residues composing the hPin1 WW three‐stranded β‐sheet structure could be replaced one at a time with at least one different natural or non‐natural amino acid residue without leading to an unfolded protein. Alanine substitutions at only four positions within the hPin1 WW domain lead to a partially or completely unfolded protein—in the absence of a physiological ligand. The side chains of these four residues form a conserved, partially solvent‐inaccessible, continuous hydrophobic minicore comprising the N‐ and C‐termini. Ala mutations at five other residues, three of which constitute the ligand binding patch on the concave side of the β‐sheet, significantly destabilize the hPin1 WW domain without leading to an unfolded protein. The remaining mutations affect protein stability only slightly, suggesting that only a small subset of side chain interactions within the hPin1 WW domain are mandatory for acquiring and maintaining a stable, cooperatively folded β‐sheet structure.     

Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope.

The single mutation L30 K in the Hu-Yap65 WW domain increased the stability of the complex with the peptide GTPPPPYTVG (K(d)=40(+/-5) microM). Here we report the refined solution structure of this complex by NMR spectroscopy and further derived structure-activity relationships by using ligand peptide libraries with truncated sequences and a substitution analysis that yielded acetyl-PPPPY as the smallest high-affinity binding peptide (K(d)=60 microM). The structures of two new complexes with weaker binding ligands chosen based on these results (N-(n-octyl)-GPPPYNH(2) and Ac-PLPPY) comprising the wild-type WW domain of Hu-Yap65 were determined. Comparison of the structures of the three complexes were useful for identifying the molecular basis of high-affinity: hydrophobic and specific interactions between the side-chains of Y28 and W39 and P5' and P4', respectively, and hydrogen bonds between T37 (donnor) and P5' (acceptor) and between W39 (donnor) and T2' (acceptor) stabilize the complex.The structure of the complex L30 K Hu-Yap65 WW domain/GTPPPPYTVG is compared to the published crystal structure of the dystrophin WW domain bound to a segment of the beta-dystroglycan protein and to the solution structure of the first Nedd4 WW domain and its prolin-rich ligand, suggesting that WW sequences bind proline-rich peptides in an evolutionary conserved fashion. The position equivalent to T22 in the Hu-Yap65 WW domain sequence is seen as responsible for differentiation in the binding mode among the WW domains of group I.     

Role of an electrostatic network of residues in the enzymatic action of the Rhizomucor miehei lipase family

We have used continuum electrostatic methods to investigate the role of electrostatic interactions in the structure, function, and pH-dependent stability of the fungal Rhizomucor miehei lipase (RmL) family. We identify a functionally important electrostatic network which includes residues S144, D203, H257, Y260, H143, Y28, R80, and D91 (residue numbering is from RmL). This network consists of residues belonging to the catalytic triad (S144, D203, H257), residues located in proximity to the active site (Y260), residues stabilizing the geometry of the active site (Y28, H143), and residues located in the lid (D91) or close to the first hinge (R80). The lid and the first hinge are associated with the interfacial activation of lipases, where an alpha-helical lid opens up by rotating around two hinge regions. All network residues are well conserved in a set of 12 lipase homologues, and 6 of the network residues are located in sequence motifs. We observe that the effects of modeled mutations R86L, D91N, and H257F on the pH-dependent electrostatic free energies differ significantly in the closed and open conformations of RmL. Mutation R86L is especially interesting since it stabilizes the closed conformation but destabilizes the open one. Site-site electrostatic interaction energies reveal that interactions between R86 and D61, D113, and E117 stabilize the open conformation.     

Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase

Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a secondsite suppressor.     

Mutational analysis of the uracil DNA glycosylase inhibitor protein and its interaction with Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase inhibitor (Ugi), a protein of 9.4 kDa consists of a five-stranded antiparallel beta sheet flanked on either side by single alpha helices, forms an exclusive complex with uracil DNA glycosylases (UDGs) that is stable in 8M urea. We report on the mutational analysis of various structural elements in Ugi, two of which (hydrophobic pocket and the beta1 edge) establish key interactions with Escherichia coli UDG. The point mutations in helix alpha1 (amino acid residues 3-14) do not affect the stability of the UDG-Ugi complexes in urea. And, while the complex of the deltaN13 mutant with UDG is stable in only approximately 4M urea, its overall structure and thermostability are maintained. The identity of P37, stacked between P26 and W68, was not important for the maintenance of the hydrophobic pocket or for the stability of the complex. However, the M24K mutation at the rim of the hydrophobic pocket lowered the stability of the complex in 6M urea. On the other hand, non-conservative mutations E49G, D61G (cancels the only ionic interaction with UDG) and N76K, in three of the loops connecting the beta strands, conferred no such phenotype. The L23R and S21P mutations (beta1 edge) at the UDG-Ugi interface, and the N35D mutation far from the interface resulted in poor stability of the complex. However, the stability of the complexes was restored in the L23A, S21T and N35A mutations. These analyses and the studies on the exchange of Ugi mutants in preformed complexes with the substrate or the native Ugi have provided insights into the two-step mechanism of UDG-Ugi complex formation. Finally, we discuss the application of the Ugi isolates in overproduction of UDG mutants, toxic to cells.     

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X-ray crystallography.

The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 A resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.     

Understanding the mechanism of beta-sheet folding from a chemical and biological perspective

Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein. Traditional site-directed mutagenesis experiments are excellent for altering side-chain structure, whereas amide-to-ester backbone mutagenesis experiments modify backbone-backbone hydrogen bonding capacity. The transition state structure associated with the folding of the Pin1 WW domain features a partially H-bonded, near-native reverse turn secondary structure in loop 1 that has little influence on thermodynamic stability. The thermodynamic stability of the Pin1 WW domain is largely determined by the formation of a small hydrophobic core and by the formation of desolvated backbone-backbone H-bonds enveloped by this hydrophobic core. Loop 1 engineering to the consensus five-residue beta-bulge-turn found in most WW domains or a four-residue beta-turn found in most beta-hairpins accelerates folding substantially relative to the six-residue turn found in the wild type Pin1 WW domain. Furthermore, the more efficient five- and four-residue reverse turns now contribute to the stability of the three-stranded beta-sheet. These insights have allowed the design of Pin1 WW domains that fold at rates that approach the theoretical speed limit of folding.     

A computational method to characterize the missense mutations in the catalytic domain of GAA protein causing Pompe disease

Pompe disease is an autosomal recessive lysosomal storage disease caused by acid α-glucosidase (GAA) deficiency, resulting in intralysosomal accumulation of glycogen, including cardiac, skeletal, and smooth muscle cells. The GAA gene is located on chromosome 17 (17q25.3), the GAA protein consists of 952 amino acids; of which 378 amino acids (347-726) falls within the catalytic domain of the protein and comprises of active sites (518 and 521) and binding sites (404, 600, 616, and 674). In this study, we used several computational tools to classify the missense mutations in the catalytic domain of GAA for their pathogenicity and stability. Eight missense mutations (R437C, G478R, N573H, Y575S, G605D, V642D, L705P, and L712P) were predicted to be pathogenic and destabilizing to the protein structure. These mutations were further subjected to phenotyping analysis using SNPeffect 4.0 to predict the chaperone binding sites and structural stability of the protein. The mutations R437C and G478R were found to compromise the chaperone-binding activity with GAA. Molecular docking analysis revealed that the G478R mutation to be more significant and hinders binding to the DNJ (Miglustat) compared with the R437C. Further molecular dynamic analysis for the two mutations demonstrated that the G478R mutation was acquired higher deviation, fluctuation, and lower compactness with decreased intramolecular hydrogen bonds compared to the mutant R437C. These data are expected to serve as a platform for drug design against Pompe disease and will serve as an ultimate tool for variant classification and interpretations.     

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C‐terminal domain

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Influence of hPin1 WW N-terminal domain boundaries on function, protein stability, and folding

An N-terminally truncated and cooperatively folded version (residues 6-39) of the human Pin1 WW domain (hPin1 WW hereafter) has served as an excellent model system for understanding triple-stranded beta-sheet folding energetics. Here we report that the negatively charged N-terminal sequence (Met1-Ala-Asp-Glu-Glu5) previously deleted, and which is not conserved in highly homologous WW domain family members from yeast or certain fungi, significantly increases the stability of hPin1 WW (approximately 4 kJ mol(-1) at 65 degrees C), in the context of the 1-39 sequence based on equilibrium measurements. N-terminal truncations and mutations in conjunction with a double mutant cycle analysis and a recently published high-resolution X-ray structure of the hPin1 cis/trans-isomerase suggest that the increase in stability is due to an energetically favorable ionic interaction between the negatively charged side chains in the N terminus of full-length hPin1 WW and the positively charged epsilon-ammonium group of residue Lys13 in beta-strand 1. Our data therefore suggest that the ionic interaction between Lys13 and the charged N terminus is the optimal solution for enhanced stability without compromising function, as ascertained by ligand binding studies. Kinetic laser temperature-jump relaxation studies reveal that this stabilizing interaction has not formed to a significant extent in the folding transition state at near physiological temperature, suggesting a differential contribution of the negatively charged N-terminal sequence to protein stability and folding rate. As neither the N-terminal sequence nor Lys13 are highly conserved among WW domains, our data further suggest that caution must be exercised when selecting domain boundaries for WW domains for structural, functional, or thermodynamic studies.     

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.     

Beta-sheet folding mechanisms from perturbation energetics

Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of beta-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in beta-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state. Energetic data and sequence mining reveal that only a small subset of the molecular information contained in sequences or observed in high-resolution structures is required to generate folded functional beta-sheets, consistent with evolutionary robustness.     

Thermal unfolding used as a probe to characterize the intra- and intersubunit stabilizing interactions in phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three nonequivalent interfaces. To investigate the contribution of intra- and intersubunit interactions to GAPDH thermostability, 10 residues located either at the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic domain (amino acids 149-312) were mutated and the thermal unfolding of the mutants was studied by differential scanning calorimetry in the absence and presence of saturating concentrations of NAD. Disruptions of intrasubunit interactions lead to a drastic decrease in thermostability of the N313T, Y283V, and W310F mutants. Moreover, for the N313T mutant, a weakening of cooperative interactions between the catalytic and the cofactor domains and an inefficient binding of NAD are observed. This is likely the consequences of modification or loss of the hydrogen bonding network associating N313 and residues 236-238 and N313 and the nicotinamide carboxyamide of NAD, respectively. For the residues Y283 and W310, which are involved in stacking hydrophobic interactions, mutating both positions does not affect the efficiency of NAD binding. This shows that the factors involved in the thermostability of the tetrameric apo GAPDH are then different from those induced by NAD binding. Disruption of intersubunit hydrogen bonds between the catalytic domain and the NAD-binding domain of a neighboring subunit also leads to a significant destabilization of the apo tetrameric form as observed for the D282G mutant. Moreover, no efficient binding of NAD is observed. Both results are likely the consequence of a loss of hydrogen bonds across the P-axis and the Q-axis between D282 and R197 and between D282 and R52, respectively. Similar results, i.e., a destabilizing effect and inefficient NAD binding, are observed with the T34Q/T39S/L43Q mutant in which steric hindrance is introduced at the S-loop of the R-axis-related subunit via mutations at the adenosine subsite. The dimeric form of the D282G mutant exhibits a single partial heat absorption peak, whereas the Y46G/R52G mutant which exists only as a dimer shows two peaks. Taking into account the recent small-angle X-ray scattering studies which suggested that the dimeric form of the D282G mutant and of the dimeric Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette, unpublished results), we propose that the presence of one or two peaks in thermal unfolding of dimers is a signature of the dimer type.     

A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues

The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a beta-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-alpha (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins.     

Molecular dissection of the CD2-CD58 counter-receptor interface identifies CD2 Tyr86 and CD58 Lys34 residues as the functional "hot spot"

The heterophilic CD2-CD58 adhesion interface contains interdigitating residues that impart high specificity and rapid binding kinetics. To define the hot spot of this counter-receptor interaction, we characterized CD2 adhesion domain variants harboring a single mutation of the central Tyr86 or of each amino acid residue forming a salt link/hydrogen bond. Alanine mutations at D31, D32 and K34 on the C strand and K43 and R48 on the C' strand reduce affinity for CD58 by 47-127-fold as measured by isothermal titration calorimetry. The Y86A mutant reduces affinity by approximately 1000-fold, whereas Y86F is virtually without effect, underscoring the importance of the phenyl ring rather than the hydroxyl moiety. The CD2-CD58 crystal structure offers a detailed view of this key functional epitope: CD2 D31 and D32 orient the side-chain of CD58 K34 such that CD2 Y86 makes hydrophobic contact with the extended aliphatic component of CD58 K34 between CD2 Y86 and CD58 F46. The elucidation of this hot spot provides a new target for rational design of immunosuppressive compounds and suggests a general approach for other receptors.     

Characterization of SH2D1A missense mutations identified in X-linked lymphoproliferative disease patients

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency characterized by extreme susceptibility to Epstein-Barr virus. The XLP disease gene product SH2D1A (SAP) interacts via its SH2 domain with a motif (TIYXXV) present in the cytoplasmic tail of the cell-surface receptors CD150/SLAM, CD84, CD229/Ly-9, and CD244/2B4. Characteristically, the SH2D1A three-pronged interaction with Tyr(281) of CD150 can occur in absence of phosphorylation. Here we analyze the effect of SH2D1A protein missense mutations identified in 10 XLP families. Two sets of mutants were found: (i) mutants with a marked decreased protein half-life (e.g. Y7C, S28R, Q99P, P101L, V102G, and X129R) and (ii) mutants with structural changes that differently affect the interaction with the four receptors. In the second group, mutations that disrupt the interaction between the SH2D1A hydrophobic cleft and Val +3 of its binding motif (e.g. T68I) and mutations that interfere with the SH2D1A phosphotyrosine-binding pocket (e.g. C42W) abrogated SH2D1A binding to all four receptors. Surprisingly, a mutation in SH2D1A able to interfere with Thr -2 of the CD150 binding motif (mutant T53I) severely impaired non-phosphotyrosine interactions while preserving unaffected the binding of SH2D1A to phosphorylated CD150. Mutant T53I, however, did not bind to CD229 and CD224, suggesting that SH2D1A controls several critical signaling pathways in T and natural killer cells. Because no correlation is present between identified types of mutations and XLP patient clinical presentation, additional unidentified genetic or environmental factors must play a strong role in XLP disease manifestations.     

Impact of autosomal recessive juvenile Parkinson's disease mutations on the structure and interactions of the parkin ubiquitin-like domain

Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson's disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP.