The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista ...structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.
PKA: Lessons learned after twenty years Taylor, Susan S.; Zhang, Ping; Steichen, Jon M. ...
Biochimica et biophysica acta,
07/2013, Letnik:
1834, Številka:
7
Journal Article
Recenzirano
Odprti dostop
The first protein kinase structure, solved in 1991, revealed the fold that is shared by all members of the eukaryotic protein kinase superfamily and showed how the conserved sequence motifs cluster ...mostly around the active site. This structure of the PKA catalytic (C) subunit showed also how a single phosphate integrated the entire molecule. Since then the EPKs have become a major drug target, second only to the G-protein coupled receptors. Although PKA provided a mechanistic understanding of catalysis that continues to serve as a prototype for the family, by comparing many active and inactive kinases we subsequently discovered a hydrophobic spine architecture that is a characteristic feature of all active kinases. The ways in which the regulatory spine is dynamically assembled is the defining feature of each protein kinase. Protein kinases have thus evolved to be molecular switches, like the G-proteins, and unlike metabolic enzymes which have evolved to be efficient catalysis. PKA also shows how the dynamic tails surround the core and serve as essential regulatory elements. The phosphorylation sites in PKA, introduced both co- and post-translationally, are very stable. The resulting C-subunit is then packaged as an inhibited holoenzyme with cAMP-binding regulatory (R) subunits so that PKA activity is regulated exclusively by cAMP, not by the dynamic turnover of an activation loop phosphate. We could not understand activation and inhibition without seeing structures of R:C complexes; however, to appreciate the structural uniqueness of each R2:C2 holoenzyme required solving structures of tetrameric holoenzymes. It is these tetrameric holoenzymes that are localized to discrete sites in the cell, typically by A Kinase Anchoring Proteins where they create discrete foci for PKA signaling. Understanding these dynamic macromolecular complexes is the challenge that we now face. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
•We discovered a hydrophobic spine architecture in protein kinases.•This architecture defines highly dynamic nature of protein kinases.•We report diverse quaternary structures for different isoforms of protein kinase A.
Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. ...Using NMR, X-ray crystallography and thermodynamic measurements, we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously and priming it for catalysis. The formation of the ternary complex is entropically driven, and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicates that a conformational selection rather than an induced-fit mechanism governs substrate recognition.
Signaling through dynamic linkers as revealed by PKA Akimoto, Madoka; Selvaratnam, Rajeevan; McNicholl, E. Tyler ...
Proceedings of the National Academy of Sciences,
08/2013, Letnik:
110, Številka:
35
Journal Article
Recenzirano
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Protein kinase A (PKA) is a prototype of multidomain signaling proteins functioning as allosteric conformational switches. Allosteric transitions have been the subject of extensive structural and ...dynamic investigations focusing mainly on folded domains. However, the current understanding of the allosteric role of partially unstructured linkers flanking globular domains is limited. Here, we show that a dynamic linker in the regulatory subunit (R) of PKA serves not only as a passive covalent thread, but also as an active allosteric element that controls activation of the kinase subunit (C) by tuning the inhibitory preequilibrium of a minimally populated intermediate (apo R). Apo R samples both C-binding competent (inactive) and incompetent (active) conformations within a nearly degenerate free-energy landscape and such degeneracy maximally amplifies the response to weak (∼2RT), but conformation-selective interactions elicited by the linker. Specifically, the R linker that in the R:C complex docks in the active site of C in apo R preferentially interacts with the C-binding incompetent state of the adjacent cAMP-binding domain (CBD). These unanticipated findings imply that the formation of the intermolecular R:C inhibitory interface occurs at the expense of destabilizing the intramolecular linker/CBD interactions in R. A direct implication of this model, which was not predictable solely based on protein structure, is that the disruption of a linker/CBD salt bridge in the R:C complex unexpectedly leads to increased affinity of R for C. The linker includes therefore sites of R:C complex frustration and frustration-relieving mutations enhance the kinase inhibitory potency of R without compromising its specificity.
Calculation of centralities in protein kinase A Kornev, Alexandr P; Aoto, Phillip C; Taylor, Susan S
Proceedings of the National Academy of Sciences - PNAS,
11/2022, Letnik:
119, Številka:
47
Journal Article
Recenzirano
Odprti dostop
Topological analysis of protein residue networks (PRNs) is a common method that can help to understand the roles of individual residues. Here, we used protein kinase A as a study object and asked ...what already known functionally important residues can be detected by network analysis. Along several traditional approaches to weight edges in PRNs we used local spatial pattern (LSP) alignment that assigns high weights to edges only if CαCβ vectors for the corresponding residues retain their mutual positions and orientation. Our results show that even short molecular dynamic simulations of 10 to 20 ns can give convergent values for betweenness and degree centralities calculated from the LSP-based PRNs. Using these centralities, we were able to clearly distinguish a group of residues that are highly conserved in protein kinases and play important functional and regulatory roles. In comparison, traditional methods based on cross-correlation and linear mutual information were much less efficient for this particular task. These results call for reevaluation of the current methods to generate PRNs.
The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the ...mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.
Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling ...scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.
Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a ...comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co‐discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by ...cAMP‐dependent protein kinase (PKA), but also by the heat‐stable, high‐affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N‐terminus a pseudo‐substrate motif that binds synergistically and with high‐affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C‐subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high‐affinity binding. The dynamic features of full‐length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C‐subunit. The type I PKA regulatory (R) subunits also contain a pseudo‐substrate ATPMg2‐dependent high‐affinity inhibitor sequence. PKI and PKA, especially the Cβ subunit, are highly expressed in the brain, and PKI expression is also cell cycle‐dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.
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