Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca 2 ] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca 2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Time-resolved x-ray solution scattering identifies cooperative structural dynamics in the adenylate kinase enzymatic reaction.

P-type ATPase proteins maintain cellular homeostasis and uphold critical concentration gradients by ATP-driven ion transport across biological membranes. Characterization of single-cycle dynamics by time-resolved X-ray scattering techniques in solution could resolve structural intermediates not amendable to for example crystallization or cryo-electron microscopy sample preparation. To pave way for such time-resolved experiments, we used biochemical activity measurements, Attenuated Total Reflectance (ATR) and time-dependent Fourier-Transform Infra-Red (FTIR) spectroscopy to identify optimal conditions for activating a Zn -transporting Type-I ATPase from Shigella sonnei (ssZntA) at high protein concentration using caged ATP. The highest total activity was observed at a protein concentration of 25 mg/mL, at 310 K, pH 7, and required the presence of 20% (v/v) glycerol as stabilizing agent. Neither the presence of caged ATP nor increasing lipid-to-protein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal-transporting (Type-I) ATPase mutants with medical relevance.

Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole. Mutants defective in Cep63•Cep152 heterotetramer formation displayed crippled pericentriolar Cep152 organization, polo-like kinase 4 (Plk4) relocalization to the procentriole assembly site, and Plk4-mediated centriole duplication. Given that the organization of pericentriolar materials (PCM) is evolutionarily conserved, this work could serve as a model for investigating the structure and function of PCM in other species, while offering a new direction in probing the organizational defects of PCM-related human diseases.

The centrosome, a unique membrane‐less organelle that serves as the main microtubule (MT)‐organizing center in animal cells, plays a pivotal role in the orderly progression of the cell cycle. Improper function of the centrosome results in abnormal cell division and proliferation that can lead the development of various human disorders. Thus, elucidating the molecular mechanisms underlying how centrosomal scaffold proteins orchestrate to generate a micrometer‐scale architecture in the three‐dimensional intracellular space is likely a key step to determining the etiology of centrosome‐associated human diseases. We found that two long coiled‐coil proteins, Cep63 and Cep152, interact with each other to form a heterotetrameric complex that self‐assembles into a higher‐order cylindrical architecture around a centriole, a barrel‐shaped organelle buried inside the centrosome. Mechanisms underlying how Cep63 and Cep152 reach their threshold concentrations in the vast cytosolic space of a cell to trigger the self‐assembly process remains not known. Using purified recombinant proteins, here we demonstrated that Cep152 and Cep63 possess an intrinsic activity of co‐phase‐separating into matrix‐like condensates and form a nanoscale cylindrical architecture. By combining sedimentation equilibrium ultracentrifugation with interferometric scattering mass spectrometry, we further showed that the heterotetrameric building block generates octameric and hexadecameric complexes in a concentration‐dependent manner in vitro, suggesting that the cylindrical self‐assembly is formed through stepwise processes. Subsequent analyses led to the discovery of a short uncharacterized region, named self‐assembly motif (SAM) from each Cep63 and Cep152, that exhibits a unique ability to undergo density transition and cooperatively generate the cylindrical self‐assembly in vitro. Fluorescent recovery after photobleaching revealed that the self‐assembly is capable of undergoing internal rearrangement within the assembly, while dynamically exchanging its components with those in the surroundings. Thus, the SAM motifs possess a highly flexible physicochemical nature that drives the Cep63‐Cep152 heterotetrameric complex to generate biomolecular condensates and self‐organize into a higher‐order cylindrical architecture likely through the repetitive cycles of controlled interactions among its constituent components. Introduction of single missense SAM mutations found in human cancer databases crippled proper recruitment and organization of Cep63 and Cep152 in vivo, thus consequently altering the function of its downstream effector, Polo‐like kinase 4, a key regulator of centriole biogenesis. Because the architecture and function of the centrosome is evolutionarily conserved, this work may serve a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms. Furthermore, given that abnormalities in the pericentriolar architecture are tightly associated with various human disorders, such as cancers, microcephaly, and dwa...

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