Biology subjects

Garman, S. C.

Publications and source records attributed to Garman, S. C..

5 recordsLinked to original sources

bioRxiv2024

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 -galactosidase A. However, the manner by which SapB stimulates -galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to -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 -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 -galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to -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.

biochemistry↗

bioRxiv2024

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

Memory formation, fertilization, and cardiac function rely on precise Ca2+ signaling and subsequent Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation. Ca2+ sensitivity of the four CaMKII paralogs in mammals has been linked to the length of the variable linker region that is a hot spot for alternative splicing. In this study, we determined that the position of charged residues within the linker modulates the Ca2+/CaM sensitivity. We present an X-ray crystal structure of the full-length CaMKII{delta} holoenzyme consisting of domain-swapped dimers within a dodecameric complex. In this structure, the kinase domain of one subunit is docked onto the hub domain of an adjacent subunit, providing an additional interface within the holoenzyme. Mutations at the hub equatorial and lateral interfaces led to alterations in the stoichiometry of CaMKII holoenzyme as well as Ca2+/CaM sensitivity. Using molecular dynamics (MD) to compare domain-swapped to non-domain-swapped CaMKIIs, we demonstrate that the domain-swapped conformation facilitates an interaction between the calmodulin binding region and the linker region. Based on MD simulations and small-angle X-ray scattering (SAXS) measurements, we propose a model where the position of charges on the linker region drives an interaction with the regulatory segment that modulates the degree of autoinhibition. Finally, we use live-cell imaging to show that the activation profiles we observe in vitro are recapitulated in cells. Our findings provide a new framework for understanding allosteric regulation of CaMKII by the linker region in Ca2+-sensitive cells.

biochemistry↗

bioRxiv2023

CaMKIIalpha and CaMKIIbeta 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 CaMKII{beta} hub domain that show 14mer (3.1 [A]) and 16mer (3.4 [A]) 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 CaMKII{beta} and hubs, which highlight major differences between these highly similar domains. We created a dimeric CaMKII{beta} 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 CaMKII{beta} hub stoichiometries are present in solution and that larger oligomers are more stable. CaMKII had a narrower distribution of molecular weight and was distinctly more stable than CaMKII{beta}.

biochemistry↗

bioRxiv2020

CaMKII binds both substrates and effectors at the active site

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. Once activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A longstanding model invokes two distinct S- and T-sites. If an interactor binds at the T-site, 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 that persistent CaMKII activity is facilitated by high affinity binding partners, which kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.

biochemistry↗

bioRxiv2017

The molecular basis for Pompe disease revealed by the structure of human acid α-glucosidase

Pompe disease results from a defect in human acid -glucosidase (GAA), a lysosomal enzyme that cleaves terminal 1-4 and 1-6 glucose from glycogen. In Pompe disease (also known as Glycogen Storage Disorder type II), the accumulation of undegraded glycogen in lysosomes leads to cellular dysfunction, primarily in muscle and heart tissues. Pompe disease is an active candidate of clinical research, with pharmacological chaperone therapy tested and enzyme replacement therapy approved. Despite production of large amounts of recombinant GAA annually, the structure of GAA has not been reported until now. Here, we describe the first structure of GAA, at 1.7[A] resolution. Three structures of GAA complexes reveal the molecular basis for the hundreds of mutations that lead to Pompe disease and for pharmacological chaperoning in the protein. The GAA structure reveals a surprising second sugar-binding site 34[A] from the active site, suggesting a possible mechanism for processing of large glycogen substrates. Overall, the structure will assist in the design of next-generation treatments for Pompe disease.

biochemistry↗