Fabry disease is an X-linked lysosomal storage disease afflicting 1 in 40,000 males with chronic pain, vascular degeneration, cardiac impairment, and other symptoms. Deficiency in the lysosomal ...enzyme α-galactosidase (α-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human α-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (β/α)
8 domain with the active site and an antiparallel β domain. N-linked carbohydrate appears at six sites in the glycoprotein dimer, revealing the basis for lysosomal transport
via the mannose-6-phosphate receptor. To understand how the enzyme cleaves galactose from glycoproteins and glycolipids, we also determined the structure of the complex of α-GAL with its catalytic product. The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively. As a point mutation in α-GAL can lead to Fabry disease, we have catalogued and plotted the locations of 245 missense and nonsense mutations in the three-dimensional structure. The structure of human α-GAL brings Fabry disease into the realm of molecular diseases, where insights into the structural basis of the disease phenotypes might help guide the clinical treatment of patients.
The majority of proteins that traverse the secretory pathway receive asparagine (Asn)-linked glycosylations. Glycans are bulky hydrophilic modifications that serve a variety of structural and ...functional roles within the cell. Here, we review the recent growing knowledge on the role of Asn-linked glycans as maturation and quality-control protein tags in the early secretory pathway. The carbohydrate composition encodes crucial information about the structure, localization and age of glycoproteins. The ‘glycan code’ is encoded by a series of glycosidases and carbohydrate transferases that line the secretory pathway. This code is deciphered by carbohydrate-binding proteins that possess distinct carbohydrate binding properties and act as molecular chaperones or sorting receptors. These glycosidases and transferases work in concert with resident secretory pathway carbohydrate-binding proteins to form a network that assists in the maturation and trafficking of both native and aberrant glycoproteins within the cell.
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. When activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition ...to the well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A long-standing model invokes two distinct S and T sites. If an interactor binds at the T-site, then it will preclude autoinhibition and allow substrates to be phosphorylated at the S site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model where persistent CaMKII activity is facilitated by high-affinity binding partners that kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.
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•CaMKII kinase domain binds all interactors through a single continuous binding site•A salt bridge interaction far from active site increases binding affinity•αD helix changes conformation between autoinhibited and active “on” states•High-affinity binders may prolong activation by maintaining the “on” state
Özden et al. report high-resolution structures of CaMKII bound to peptides from proteins that are crucial for memory. All interactors dock onto the same single binding site using conserved interactions. A model is proposed for how high-affinity interactors prolong CaMKII activity by competing with autoinhibition.
Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been ...linked to Niemann-Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.
Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α- N -acetylgalactosaminidase (α-NAGAL) ...enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo. We demonstrate that a related iminosugar DGJ (currently in phase III clinical trials for another metabolic disorder, Fabry disease) can also chaperone human α-NAGAL in Schindler/Kanzaki disease. The 1.4- and 1.5-Å crystal structures of human α-NAGAL complexes reveal the different binding modes of iminosugars compared with glycosides. We show how differences in two functional groups result in >9 kcal/mol of additional binding energy and explain the molecular interactions responsible for the unexpectedly high affinity of the pharmacological chaperones. These results open two avenues for treatment of Schindler/Kanzaki disease and elucidate the atomic basis for pharmacological chaperoning in the entire family of lysosomal storage diseases.
Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. ...The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) is deficient in patients with the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A). Here, we report the three-dimensional structure of human GALNS, determined by X-ray crystallography at 2.2Å resolution. The structure reveals a catalytic gem diol nucleophile derived from modification of a cysteine side chain. The active site of GALNS is a large, positively charged trench suitable for binding polyanionic substrates such as keratan sulfate and chondroitin-6-sulfate. Enzymatic assays on the insect‐cell-expressed human GALNS indicate activity against synthetic substrates and inhibition by both substrate and product. Mapping 120 MPS IV A missense mutations onto the structure reveals that a majority of mutations affect the hydrophobic core of the structure, indicating that most MPS IV A cases result from misfolding of GALNS. Comparison of the structure of GALNS to paralogous sulfatases shows a wide variety of active‐site geometries in the family but strict conservation of the catalytic machinery. Overall, the structure and the known mutations establish the molecular basis for MPS IV A and for the larger MPS family of diseases.
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► We have determined the crystal structure of human GALNS. ► The structure reveals the molecular basis of MPS IV A (Morquio A) disease. ► The structure shows posttranslational modification of an active‐site cysteine. ► The structure reveals that MPS IV A (Morquio A) is a protein-folding disease. ► Human GALNS is a promising target for rational drug design.
Proteolytic Activation of Human Cathepsin A Kolli, Nilima; Garman, Scott C.
Journal of biological chemistry/The Journal of biological chemistry,
04/2014, Letnik:
289, Številka:
17
Journal Article
Recenzirano
Odprti dostop
Galactosialidosis is a human lysosomal storage disease caused by deficiency in the multifunctional lysosomal protease cathepsin A (also known as protective protein/cathepsin A, PPCA, catA, HPP, and ...CTSA; EC 3.4.16.5). Previous structural work on the inactive precursor human cathepsin A (zymogen) led to a two-stage model for activation, where proteolysis of a 1.6-kDa excision peptide is followed by a conformational change in a blocking peptide occluding the active site. Here we present evidence for an alternate model of activation of human cathepsin A, needing only cleavage of a 3.3-kDa excision peptide to yield full enzymatic activity, with no conformational change required. We present x-ray crystallographic, mass spectrometric, amino acid sequencing, enzymatic, and cellular data to support the cleavage-only activation model. The results clarify a longstanding question about the mechanism of cathepsin A activation and point to new avenues for the design of mechanism-based inhibitors of the enzyme.
The human lysosomal protease cathepsin A requires proteolytic activation.
The crystal structure of mature and active cathepsin A reveals its mechanism of activation.
Removal of a 3.3-kDa peptide (by unidentified proteases) allows substrate access to the active site.
The results revise a 2-decades-old model of cathepsin A activation by proteolysis and subsequent conformational change.
Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a multidomain serine/threonine kinase that plays important roles in the brain, heart, muscle tissue, and eggs/sperm. The N‐terminal kinase and ...regulatory domain is connected by a flexible linker to the C‐terminal hub domain. The hub domain drives the oligomeric organization of CaMKII, assembling the kinase domains into high local concentration. Previous structural studies have shown multiple stoichiometries of the holoenzyme as well as the hub domain alone. Here, we report a comprehensive study of the hub domain stoichiometry and stability in solution. We solved two crystal structures of the CaMKIIβ hub domain that show 14‐mer (3.1 Å) and 16‐mer (3.4 Å) assemblies. Both crystal structures were determined from crystals grown in the same drop, which suggests that CaMKII oligomers with different stoichiometries likely coexist. To further interrogate hub stability, we employed mass photometry and temperature denaturation studies of CaMKIIβ and CaMKIIα hubs, which highlight major differences between these highly similar domains. We created a dimeric CaMKIIβ hub unit using rational mutagenesis, which is significantly less stable than the oligomer. Both hub domains populate an intermediate during unfolding. We found that multiple CaMKIIβ hub stoichiometries are present in solution and that larger oligomers are more stable. CaMKIIα had a narrower distribution of molecular weight and was distinctly more stable than CaMKIIβ.
Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A ...deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.
A polymeric nanogel has been used to sequester and turn off a lysosomal protein, acid α-glucosidase (GAA). The nanogel contains a β-thiopropionate cross-linker, which endows the nanogel with ...pH-sensitivity. While encapsulation of the enzyme fully turns off its activity, approximately 75% of the activity is recovered upon reducing the pH to 5.0. The recovered activity is ascribed to pH-induced degradation of the β-thiopropionate cross-linker causing the swelling of the nanogel and ultimately causing the release of the enzyme. We envision that strategies for sequestering protein molecules and releasing them at lysosomal pH might open up new directions for therapeutic treatment of lysosomal storage diseases.
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