A domain-swapped CaMKII conformation facilitates linker-mediated allosteric regulation.

Ca2+ signaling plays a key role in physiological processes such as memory formation and cardiac function. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the primary kinase that responds to Ca2+ inputs in these cells. There are four CaMKII paralogs in mammals which are alternatively spliced in the variable linker region to create upwards of 70 different variants. In this study, we systematically studied different linker regions and determined that the position of charged residues within the linker region modulates the Ca2+/CaM sensitivity of the holoenzyme. We present an X-ray crystal structure of full-length CaMKIIδ that shows a domain-swapped conformation of the subunits within the dodecameric holoenzyme. In this structure, the kinase domain of one subunit is docked onto the hub domain of a different subunit, providing an additional interface within the holoenzyme. Mutations at the equatorial and lateral interfaces revealed that the kinase-hub interaction dissociates as the hub-hub interfaces are disturbed, which led alterations in the stoichiometry of CaMKII holoenzyme and Ca2+/CaM sensitivity. Molecular dynamics simulations of linker-containing domain-swapped and non-domain-swapped CaMKIIs reveal that the domain-swapped configuration facilitates an interaction between the calmodulin binding domain and the variable linker region, such that dynamic electrostatic forces between charges on these segments can modulate the equilibrium between the compact and extended conformational states of the holoenzyme. Small angle X-ray scattering data confirms that a negatively charged linker CaMKII holoenzyme adopts a more compact conformation compared to a positively charged linker. These data support a model where patches of charged linker residues interact with the calmodulin binding domain to allosterically regulate sensitivity to Ca2+/CaM. Our findings provide a new framework for understanding CaMKII structure and allosteric regulation by the variable linker region in Ca2+-sensitive cells.

Human Saposin B Ligand Binding and Presentation to α-Galactosidase A.

Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates α-galactosidase A cleavage of Gb3-NBD in vitro. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactions between SapB and α-galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to α-galactosidase A. These findings establish general principles for molecular recognition in saposin:hydrolase complexes and highlight the utility of NBD reporter lipids in saposin biochemistry and structural biology.

Ca2+/CaM dependent protein kinase II (CaMKII)α and CaMKIIß hub domains adopt distinct oligomeric states and stabilities.

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 CaMKIIß 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 CaMKIIß and CaMKIIα hubs, which highlight major differences between these highly similar domains. We created a dimeric CaMKIIß 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 CaMKIIß 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 CaMKIIß.

CaMKII binds both substrates and activators at the active site.

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.

Characterization of CaMKIIα holoenzyme stability.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca2+ -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent Tm = 36°C), which is slightly stabilized by ATP/MgCl2 binding (apparent Tm = 40°C) and significantly stabilized by regulatory segment binding (apparent Tm = 60°C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent Tm ~ 90°C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60°C to 100°C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact.

Phenotypic characteristics of the p.Asn215Ser (p.N215S) GLA mutation in male and female patients with Fabry disease: A multicenter Fabry Registry study.

BACKGROUND: The p.Asn215Ser or p.N215S GLA variant has been associated with late-onset cardiac variant of Fabry disease. METHODS: To expand on the scarce phenotype data, we analyzed natural history data from 125 p.N215S patients (66 females, 59 males) enrolled in the Fabry Registry (NCT00196742) and compared it with data from 401 patients (237 females, 164 males) harboring mutations associated with classic Fabry disease. We evaluated interventricular septum thickness (IVST), left ventricular posterior wall thickness (LVPWT), estimated glomerular filtration rate and severe clinical events. RESULTS: In p.N215S males, mildly abnormal mean IVST and LVPWT values were observed in patients aged 25-34 years, and values gradually increased with advancing age. Mean values were similar to those of classic males. In p.N215S females, these abnormalities occurred primarily in patients aged 55-64 years. Severe clinical events in p.N215S patients were mainly cardiac (males 31%, females 8%) while renal and cerebrovascular events were rare. Renal impairment occurred in 17% of p.N215S males (mostly in patients aged 65-74 years), and rarely in females (3%). CONCLUSION: p.N215S is a disease-causing mutation with severe clinical manifestations found primarily in the heart. Cardiac involvement may become as severe as in classic Fabry patients, especially in males.

Ligand-promoted protein folding by biased kinetic partitioning.

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

Human acid sphingomyelinase structures provide insight to molecular basis of Niemann-Pick disease.

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.

Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.

Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the NH group of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intrasubunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network and compare the results with prior predictions from density functional theory calculations. The X-ray crystal structure of H1A-NiSOD, which lacks the apical ligand entirely, reveals that in the absence of the Glu17-His1 H-bond, the active site is disordered. Characterization of this variant using X-ray absorption spectroscopy (XAS) shows that Ni(II) is bound in the expected N2S2 planar coordination site. Despite these structural perturbations, the H1A-NiSOD variant retains 4% of wild-type (WT) NiSOD activity. Three other mutations were designed to preserve the apical imidazole ligand but perturb the H-bonding network: R47A-NiSOD, which lacks the intramolecular H-bonding interaction; E17R/R47A-NiSOD, which retains the intramolecular H-bond but lacks the intermolecular Glu17-His1 H-bond; and E17A/R47A-NiSOD, which lacks both H-bonding interactions. These variants were characterized by a combination of techniques, including XAS to probe the nickel site structure, kinetic studies employing pulse-radiolytic production of superoxide, and electron paramagnetic resonance to assess the Ni redox activity. The results indicate that in addition to the roles in redox tuning suggested on the basis of previous computational studies, the Glu17-His1 H-bond plays an important structural role in the proper folding of the "Ni-hook" motif that is a critical feature of the active site.

The alpha-galactosidase A p.Arg118Cys variant does not cause a Fabry disease phenotype: data from individual patients and family studies.

Lysosomal α-galactosidase A (α-Gal) is the enzyme deficient in Fabry disease (FD), an X-linked glycosphingolipidosis caused by pathogenic mutations affecting the GLA gene. The early-onset, multi-systemic FD classical phenotype is associated with absent or severe enzyme deficiency, as measured by in vitro assays, but patients with higher levels of residual α-Gal activity may have later-onset, more organ-restricted clinical presentations. A change in the codon 118 of the wild-type α-Gal sequence, replacing basic arginine by a potentially sulfhydryl-binding cysteine residue - GLA p.(Arg118Cys) -, has been recurrently described in large FD screening studies of high-risk patients. Although the Cys118 allele is associated with high residual α-Gal activity in vitro, it has been classified as a pathogenic mutation, mainly on the basis of theoretical arguments about the chemistry of the cysteine residue. However its pathogenicity has never been convincingly demonstrated by pathology criteria. We reviewed the clinical, biochemical and histopathology data obtained from 22 individuals of Portuguese and Spanish ancestry carrying the Cys118 allele, including 3 homozygous females. Cases were identified either on the differential diagnosis of possible FD manifestations and on case-finding studies (n=11; 4 males), or on unbiased cascade screening of probands' close relatives (n=11; 3 males). Overall, those data strongly suggest that the GLA p.(Arg118Cys) variant does not segregate with FD clinical phenotypes in a Mendelian fashion, but might be a modulator of the multifactorial risk of cerebrovascular disease. The Cys118 allelic frequency in healthy Portuguese adults (n=696) has been estimated as 0.001, therefore not qualifying for "rare" condition.

Unlocking a caged lysosomal protein from a polymeric nanogel with a pH trigger.

A polymeric nanogel has been used to sequester and turn off a lysosomal protein, acid α-glucosidase (GAA). The nanogel contains a ß-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 ß-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.

Proteolytic activation of human cathepsin A.

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.

Electrostatic origin of in vitro aggregation of human γ-crystallin.

The proteins α-, ß-, and γ-crystallins are the major components of the lens in the human eye. Using dynamic light scattering method, we have performed in vitro investigations of protein-protein interactions in dilute solutions of human γ-crystallin and α-crystallin. We find that γ-crystallin spontaneously aggregates into finite-sized clusters in phosphate buffer solutions. There are two distinct populations of unaggregated and aggregated γ-crystallins in these solutions. On the other hand, α-crystallin molecules are not aggregated into large clusters in solutions of α-crystallin alone. When α-crystallin and γ-crystallin are mixed in phosphate buffer solutions, we demonstrate that the clusters of γ-crystallin are prevented. By further investigating the roles of temperature, protein concentration, pH, salt concentration, and a reducing agent, we show that the aggregation of γ-crystallin under our in vitro conditions arises from non-covalent electrostatic interactions. In addition, we show that aggregation of γ-crystallin occurs under the dilute in vitro conditions even in the absence of oxidizing agents that can induce disulfide cross-links, long considered to be responsible for human cataracts. Aggregation of γ-crystallin when maintained under reducing conditions suggests that oxidation does not contribute to the aggregation in dilute solutions.

Pharmacological chaperones for human α-N-acetylgalactosaminidase.

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.

The structure of human GALNS reveals the molecular basis for mucopolysaccharidosis IV A.

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.

Adaptor-dependent degradation of a cell-cycle regulator uses a unique substrate architecture.

In Caulobacter crescentus, the ClpXP protease degrades several crucial cell-cycle regulators, including the phosphodiesterase PdeA. Degradation of PdeA requires the response regulator CpdR and signals a morphological transition in concert with initiation of DNA replication. Here, we report the structure of a Per-Arnt-Sim (PAS) domain of PdeA and show that it is necessary for CpdR-dependent degradation in vivo and in vitro. CpdR acts as an adaptor, tethering the amino-terminal PAS domain to ClpXP and promoting recognition of the weak carboxyl-terminal degron of PdeA, a combination that ensures processive proteolysis. We identify sites on the PAS domain needed for CpdR recognition and find that one subunit of the PdeA dimer can be delivered to ClpXP by its partner. Finally, we show that improper stabilization of PdeA in vivo alters cellular behavior. These results introduce an adaptor/substrate pair for ClpXP and reveal broad diversity in adaptor-mediated proteolysis.

The molecular basis of pharmacological chaperoning in human α-galactosidase.

Fabry disease patients show a deficiency in the activity of the lysosomal enzyme α-galactosidase (α-GAL or α-Gal A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the α-GAL protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human α-GAL glycoprotein. Crystal structures of complexes of α-GAL and chaperones explain the molecular basis for the higher potency of DGJ over galactose. Using site-directed mutagenesis, we show the higher potency of DGJ results from an ionic interaction with D170. We propose that protonation of D170 in acidic conditions leads to weaker binding of DGJ. The results establish a biochemical basis for pharmacological chaperone therapy applicable to other protein misfolding diseases.

GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings.

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.

Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.

The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

Catalytic mechanism of human alpha-galactosidase.

The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of alpha-galactosides in the lysosome. Defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues. alpha-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human alpha-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-alpha-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a (1)S(3) skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on alpha-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.