CryoEM maps are full of potential Marques, Mayra A; Purdy, Michael D; Yeager, Mark
Current opinion in structural biology,
10/2019, Letnik:
58
Journal Article
Recenzirano
Odprti dostop
•Maps derived by X-ray crystallography display electron density, whereas maps derived by electron microscopy display Coulomb potential.•Negatively charged oxygen can result in attenuated positive ...potentials and even regions of negative density or ‘holes’ in Coulomb potential maps.•Biological molecules are exquisitely sensitive to damage from electron irradiation, especially the carboxylates of acidic amino acids.•A challenge in the interpretation of Coulomb potential maps is distinguishing the relative contributions from the charged states of oxygen atoms versus radiation damage.•High-resolution Coulomb potential maps can reveal information lacking in electron density maps, including protein and ion charged states, bond visualization and the presence of hydrogen atoms.
Electron microscopy is based on elastic scattering due to Coulomb forces between the incident electrons and the sample; thus, electron scattering is dependent on the charge distribution in the sample. Unlike atomic scattering factors for X-rays, electron scattering factors for some atoms are strongly dependent on scattering angle, and the scattering factor for ionic oxygen is negative at low scattering angle. This phenomenon can result in a significant negative contribution to Coulomb potential maps by oxygen and can result in deviations in the positions of positive map features from atomic centers. An important factor that can also complicate the interpretation of cryoEM maps is the exquisite sensitivity of macromolecules to damage from electron irradiation, especially the carboxylates of acidic amino acids. Ideally, when compared with electron density maps derived by X-ray crystallography, Coulomb potential maps can provide additional details about the electrostatic environment and charge state of atoms. Enhancements in model building, refinement and computational simulation will be required to realize the full potential of EM-derived maps to reveal deeper insight into the electronic structure and functional properties of macromolecular complexes and their interactions with binding partners, ligands, cofactors, and drugs.
Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF). The mechanism of the ...formation of the aggregates in the nucleus remains uncertain. The present study demonstrated that the DNA-binding domain of p53 (p53C) underwent phase separation (PS) on the pathway to aggregation under various conditions. p53C phase separated in the presence of the crowding agent polyethylene glycol (PEG). Similarly, mutant p53C (M237I and R249S) underwent PS; however, the process evolved to a solid-like phase transition faster than that in the case of wild-type p53C. The data obtained by microscopy of live cells indicated that transfection of mutant full-length p53 into the cells tended to result in PS and phase transition (PT) in the nuclear compartments, which are likely the cause of the GoF effects. Fluorescence recovery after photobleaching (FRAP) experiments revealed liquid characteristics of the condensates in the nucleus. Mutant p53 tended to undergo gel- and solid-like phase transitions in the nucleus and in nuclear bodies demonstrated by slow and incomplete recovery of fluorescence after photobleaching. Polyanions, such as heparin and RNA, were able to modulate PS and PT
in vitro
. Heparin apparently stabilized the condensates in a gel-like state, and RNA apparently induced a solid-like state of the protein even in the absence of PEG. Conditions that destabilize p53C into a molten globule conformation also produced liquid droplets in the absence of crowding. The disordered transactivation domain (TAD) modulated both phase separation and amyloid aggregation. In summary, our data provide mechanistic insight into the formation of p53 condensates and conditions that may result in the formation of aggregated structures, such as mutant amyloid oligomers, in cancer. The pathway of mutant p53 from liquid droplets to gel-like and solid-like (amyloid) species may be a suitable target for anticancer therapy.
Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF).
Two new complexes of Ru(II) with mixed ligands were prepared: Ru(bpy)
smp(PF
) (
) and Ru(phen)
smp(PF
) (
), in which smp = sulfamethoxypyridazine; bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline. ...The complexes have been characterized by elemental and conductivity analyses; infrared, NMR, and electrospray ionization mass spectroscopies; and X-ray diffraction of single crystal. Structural analyses reveal a distorted octahedral geometry around Ru(II) that is bound to two bpy (in
) or two phen (in
) via their two heterocyclic nitrogens and to two nitrogen atoms from sulfamethoxypyridazine-one of the methoxypyridazine ring and the sulfonamidic nitrogen, which is deprotonated. Both complexes inhibit the growth of chronic myelogenous leukemia cells. The interaction of the complexes with bovine serum albumin and DNA is described. DNA footprinting using an oligonucleotide as substrate showed the complexes' preference for thymine base rich sites. It is worth notifying that the complexes interact with the Src homology SH3 domain of the Abl tyrosine kinase protein. Abl protein is involved in signal transduction and implicated in the development of chronic myelogenous leukemia. Nuclear magnetic resonance (NMR) studies of the interaction of complex
with the Abl-SH3 domain showed that the most affected residues were T79, G97, W99, and Y115.
Biomolecular condensates, membrane-less entities arising from liquid–liquid phase separation, hold dichotomous roles in health and disease. Alongside their physiological functions, these condensates ...can transition to a solid phase, producing amyloid-like structures implicated in degenerative diseases and cancer. This review thoroughly examines the dual nature of biomolecular condensates, spotlighting their role in cancer, particularly concerning the p53 tumor suppressor. Given that over half of the malignant tumors possess mutations in the TP53 gene, this topic carries profound implications for future cancer treatment strategies. Notably, p53 not only misfolds but also forms biomolecular condensates and aggregates analogous to other protein-based amyloids, thus significantly influencing cancer progression through loss-of-function, negative dominance, and gain-of-function pathways. The exact molecular mechanisms underpinning the gain-of-function in mutant p53 remain elusive. However, cofactors like nucleic acids and glycosaminoglycans are known to be critical players in this intersection between diseases. Importantly, we reveal that molecules capable of inhibiting mutant p53 aggregation can curtail tumor proliferation and migration. Hence, targeting phase transitions to solid-like amorphous and amyloid-like states of mutant p53 offers a promising direction for innovative cancer diagnostics and therapeutics.
The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what ...triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.
Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we ...evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca2+-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC–cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.
The gene encoding the p53 tumor suppressor protein is the most frequently mutated oncogene in cancer patients; yet, generalized strategies for rescuing the function of different p53 mutants remain ...elusive. This work investigates factors that may contribute to the low inherent stability of the wild-type p53 core domain (p53C) and structurally compromised Y220C mutant. Pressure-induced unfolding of p53C was compared to p63C, the p53 family member with the highest stability, the engineered superstable p53C hexamutant (p53C HM), and lower stability p53C Y220C cancer-associated mutant. The following pressure unfolding values (P50% bar) were obtained: p53C 3346, p53C Y220C 2217, p53C HM 3943, and p63C 4326. Molecular dynamics (MD) simulations revealed that p53C Y220C was most prone to water infiltration, followed by p53C, whereas the interiors of p53C HM and p63C remained comparably dry. A strong correlation (r 2 = 0.92) between P50% and extent of interior hydration was observed. The pathways of individual water molecule entry and exit were mapped and analyzed, revealing a common route preserved across the p53 family involving a previously reported pocket, along with a novel surface cleft, both of which appear to be targetable by small molecules. Potential determinants of propensity to water incursion were assessed, including backbone hydrogen bond protection and combined sequence and structure similarity. Collectively, our results indicate that p53C has an intrinsic susceptibility to water leakage, which is exacerbated in a structural class mutant, suggesting that there may be a common avenue for rescuing p53 function.
Despite being referred to as the guardian of the genome, when impacted by mutations, p53 can lose its protective functions and become a renegade. The malignant transformation of p53 occurs on ...multiple levels, such as altered DNA binding properties, acquisition of novel cellular partners, or associating into different oligomeric states. The consequences of these transformations can be catastrophic. Ongoing studies have implicated different oligomeric p53 species as having a central role in cancer biology; however, the correlation between p53 oligomerization status and oncogenic activities in cancer progression remains an open conundrum. In this review, we summarize the roles of different p53 oligomeric states in cancer and discuss potential research directions for overcoming aberrant p53 function associated with them. We address how misfolding and prion-like amyloid aggregation of p53 seem to play a crucial role in cancer development. The misfolded and aggregated states of mutant p53 are prospective targets for the development of novel therapeutic strategies against tumoral diseases.
The p53 protein is a pleiotropic regulator working as a tumor suppressor and as an oncogene. Depending on the cellular insult and the mutational status, p53 may trigger opposing activities such as ...cell death or survival, senescence and cell cycle arrest or proliferative signals, antioxidant or prooxidant activation, glycolysis, or oxidative phosphorylation, among others. By augmenting or repressing specific target genes or directly interacting with cellular partners, p53 accomplishes a particular set of activities. The mechanism in which p53 is activated depends on increased stability through post-translational modifications (PTMs) and the formation of higher-order structures (HOS). The intricate cell death and metabolic p53 response are reviewed in light of gaining stability
via
PTM and HOS formation in health and disease.
The missing links within troponin Marques, Mayra A.; Parvatiyar, Michelle S.; Yang, Wei ...
Archives of biochemistry and biophysics,
03/2019, Letnik:
663
Journal Article
Recenzirano
Odprti dostop
The cardiac contraction-relaxation cycle is controlled by a sophisticated set of machinery. Of particular interest, is the revelation that allosteric networks transmit effects of binding at one site ...to influence troponin complex dynamics and structural-mediated signaling in often distal, functional sites in the myofilament. Our recent observations provide compelling evidence that allostery can explain the function of large-scale macromolecular events. Here we elaborate on our recent findings of interdomain communication within troponin C, using cutting-edge structural biology approaches, and highlight the importance of unveiling the unknown, distant communication networks within this system to obtain more comprehensive knowledge of how allostery impacts cardiac physiology and disease.