JMS/Sandbox/msn2 gemini 0
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Msn2: A Molecular Tour of Yeast's Master Stress Regulator - Structure, Function, and Interactive Exploration1. Msn2: Master Regulator of Yeast Stress ResponseThe budding yeast Saccharomyces cerevisiae employs a sophisticated network of regulatory proteins to adapt and survive in fluctuating and often harsh environments. Central to this adaptation is the general stress response (GSR), also known as the environmental stress response (ESR), a broad transcriptional program that fortifies the cell against a multitude of challenges. The transcription factor Msn2, along with its close paralog Msn4, stands as a master regulator of this critical defense system.1 These proteins are responsible for activating the expression of hundreds of genes, estimated to be around 200 distinct targets or influencing over 90% of genes upregulated during common stresses, thereby enabling the cell to cope with diverse adverse conditions.1Msn2/Msn4 achieve this widespread gene activation by recognizing and binding to specific DNA sequences known as Stress Response Elements (STREs) located in the promoter regions of their target genes.1 The consensus sequence for STREs is typically 5'-AGGGG or 5'-CCCCT.1 The range of environmental insults that trigger the Msn2-mediated stress response is extensive, including heat shock, osmotic stress, oxidative damage, glucose starvation, sorbic acid, high ethanol concentrations, and deviations in pH.1 This broad reactivity underscores Msn2's role as a central hub for integrating various stress signals. The ability of a single (or pair of) transcription factor to respond to such a wide array of stimuli implies the existence of numerous upstream sensory and signaling pathways that converge upon Msn2/Msn4. This architecture suggests an evolutionary advantage in having a generalized response mechanism, positioning these factors not merely as simple molecular switches but as crucial decision-making nodes that allow the cell to mount a robust defense irrespective of the specific nature of the threat.The activity of Msn2 is itself tightly regulated, primarily through its dynamic shuttling between the cytoplasm and the nucleus.1 Under favorable, non-stress conditions, Msn2 is predominantly localized in the cytoplasm, effectively keeping it sequestered from its target genes in the nucleus.1 Upon encountering stress, however, Msn2 rapidly translocates to the nucleus, where it can bind STREs and initiate the transcription of stress-protective genes. This nucleocytoplasmic trafficking is intricately controlled by major cellular signaling pathways, most notably the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway and the Target of Rapamycin (TOR) pathway.1While Msn2 and Msn4 share significant sequence homology (around 66%) and are often described as functionally redundant, evidence suggests a more nuanced relationship.1 Studies indicate that Msn2 and Msn4 can have differential contributions to the expression of specific genes or under particular stress conditions.2 This hints at a finer layer of regulatory control than a simple on/off mechanism, possibly arising from subtle differences in their activation thresholds, preferences for STRE variants, interactions with other regulatory proteins, or post-translational modifications. Msn2 appears to play a dominant role under most conditions in the ESR.5 Understanding the specific structural features of Msn2, as explored in this article, may provide clues to the basis of these potential functional distinctions from Msn4.2. The Msn2 Protein: Sequence Features and IdentifiersMsn2 from the reference strain Saccharomyces cerevisiae S288c (Systematic Name: YMR037C; SGD ID: S000004640; UniProt ID: P33748) is a protein composed of 704 amino acids.2 Its calculated molecular weight is approximately 77.85 kDa.2 Msn2 is characterized by a modular architecture, comprising distinct domains responsible for DNA binding, nuclear import and export, and transcriptional activation. These domains, along with key regulatory sites, are summarized in Table 1.Table 1: Summary of Key Msn2 Functional Regions and Sites (UniProt P33748 / SGD S000004640) Feature TypeResidue Range / Specific SitesPrimary Function/RoleKey Regulatory Aspects / NotesC2H2 Zinc Finger 1 (ZF1)~441-463 6DNA binding (STRE recognition)C-terminal region; classical C2H2 foldC2H2 Zinc Finger 2 (ZF2)~471-493 6DNA binding (STRE recognition)C-terminal region, tandem to ZF1Nuclear Localization Signal (NLS)~576-648 7Nuclear importMediates interaction with importins (e.g., Kap123 9); PKA phosphorylation (e.g., S582, S620, S625, S633) inhibits importNuclear Export Signal (NES)~237-327 (minimal region) 10Nuclear export (Msn5-dependent)Contains HD1 (aa 259-314); PKA phosphorylation (e.g., S288, S304) activates exportIntrinsically Disordered RegionN-terminal (~1-236), Linker (~328-440; ~494-575), C-term (~649-704)Transcriptional activation, co-activator binding, flexibilityContains TAD motifs (e.g., 1-50, 253-267 11); low pLDDT in AlphaFold modelsKey Phospho Site (NES)S288 10Regulates NES activity (PKA site)Phosphorylation promotes exportKey Phospho Site (NES)S304 10Regulates NES activityPhosphorylation promotes exportKey Phospho Site (NLS)S582 10Regulates NLS activity (PKA/Snf1 site)Phosphorylation inhibits importKey Phospho Site (NLS)S620 10Regulates NLS activity (PKA site)Phosphorylation inhibits importKey Phospho Site (NLS)S625 10Regulates NLS activity (PKA site)Phosphorylation inhibits importKey Phospho Site (NLS)S633 10Regulates NLS activity (PKA site)Phosphorylation inhibits importKey Phospho Site (DBD-proximal)S686 10Near DNA binding domain (PKA site)Potential role in DNA binding/activity
Note: IDR ranges are approximate based on general descriptions and specific annotations from related entries like A2Q129 13, adapted for P33748. Precise boundaries of IDRs can be fluid.The modular nature of Msn2 is evident, with distinct regions dedicated to sensing regulatory signals (via phosphorylation sites), interacting with DNA (zinc fingers), controlling its subcellular location (NLS, NES), and effecting changes in gene expression (transcriptional activation domains within IDRs). However, these modules do not operate in isolation. Their functions are tightly interwoven, particularly through post-translational modifications like phosphorylation, which can coordinately regulate NLS and NES activities to achieve precise control over Msn2's presence in the nucleus.1 The extensive intrinsically disordered regions likely provide the conformational flexibility necessary for these domains to interact with various regulators, the nuclear transport machinery, and components of the transcriptional apparatus.When examining structural models or domain annotations, it is important to consider the source and specificity of the data. For instance, predicted domain boundaries can vary slightly between different bioinformatics tools or even between annotations for different strains of the same organism. For the purpose of this molecular tour, which focuses on the AlphaFold model of Msn2 from S. cerevisiae S288c (UniProt P33748), residue ranges derived from analyses specific to this strain or well-corroborated experimental data are prioritized.6 This careful curation is essential for accurately mapping functional features onto the 3D structure.3. Molecular Tour of Msn2's 3D Structure (AlphaFold Model P33748)This section provides an interactive exploration of the three-dimensional structure of Msn2, as predicted by AlphaFold for the UniProt entry P33748. AlphaFold models have become invaluable resources for structural biology, offering high-accuracy predictions, particularly for well-ordered domains. For proteins like Msn2 that contain significant flexible or intrinsically disordered regions, AlphaFold also provides a per-residue confidence score called pLDDT (predicted local distance difference test). This score, typically stored in the B-factor column of the PDB file, ranges from 0 to 100, where higher scores indicate higher confidence in the predicted local structure.15 We will use this pLDDT score to guide our tour and interpret the structural features.
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Caption for the link above: Msn2 colored by AlphaFold pLDDT score. Blue indicates high confidence (pLDDT near 100), yellow indicates medium confidence, and red indicates low confidence (pLDDT near 0).3.2. C-Terminal C2H2 Zinc Finger Domains: The DNA GrippersLocated in the C-terminal region of Msn2 are two classical C2H2-type zinc finger domains, which are essential for its function as a transcription factor.2 For the S288c strain (P33748), these are:
Zinc Finger 1 (ZF1): Residues approximately 441-463.
Zinc Finger 2 (ZF2): Residues approximately 471-493.
These domains adopt a characteristic ββα fold, where cysteine and histidine residues coordinate a zinc ion (note: the zinc ion itself is typically not modeled by AlphaFold unless present in a template structure and explicitly modeled). This structural motif allows the zinc fingers to make specific contacts with the DNA bases and backbone of the STRE sequences (e.g., 5'-AGGGG or 5'-CCCCT) found in the promoters of Msn2 target genes.1 The tandem arrangement of these two zinc fingers likely enhances both the affinity and specificity of DNA binding. The high pLDDT scores typically observed for such well-defined domains in AlphaFold models should allow for a reliable visualization of their structure and relative orientation, providing insights into how Msn2 recognizes its DNA targets.
Caption for the link above: Msn2's C-terminal tandem C2H2 zinc finger domains (ZF1: 441-463, ZF2: 471-493), crucial for DNA binding, are shown in green (ball-and-stick) with the rest of the protein as a light grey cartoon. Labels indicate ZF1 and ZF2.3.3. Nuclear Localization Signal (NLS): Gatekeeper for Nuclear EntryAdjacent to the C-terminal zinc finger domains lies the Nuclear Localization Signal (NLS), a sequence motif responsible for mediating Msn2's import into the nucleus.2 This region has been mapped to encompass residues approximately 576-648.7 The NLS interacts with components of the nuclear import machinery, such as importin α and Kap123, facilitating Msn2's passage through the nuclear pore complex.9 Structurally, NLS sequences are often rich in basic amino acids (lysine and arginine) and can be relatively flexible to allow for effective interaction with importin proteins. The pLDDT scores in this region of the AlphaFold model will indicate how well-defined its predicted structure is.The function of the Msn2 NLS is critically regulated by phosphorylation. Several serine residues within and flanking this sequence are targets for Protein Kinase A (PKA). Phosphorylation at these sites, notably S582, S620, S625, and S633, inhibits NLS activity, thereby preventing nuclear import and retaining Msn2 in the cytoplasm under non-stress conditions.2 The concentration of these regulatory phosphorylation sites within a relatively compact NLS underscores its role as a sensitive switch controlling Msn2's access to its nuclear targets. The AlphaFold model may provide clues about the surface accessibility of these serine residues, which is a prerequisite for their modification by kinases and phosphatases.
Caption for the link above: The Nuclear Localization Signal (NLS) of Msn2 (residues 576-648), responsible for nuclear import, is highlighted in magenta cartoon representation.3.4. Nuclear Export Signal (NES): Facilitating Cytoplasmic ReturnComplementary to the NLS, Msn2 possesses a Nuclear Export Signal (NES) that mediates its removal from the nucleus, ensuring its return to the cytoplasm when stress conditions subside or under basal conditions.2 This NES is located within the N-terminal half of the protein, with a minimal functional region identified as residues approximately 237-327.10 This region notably includes a conserved segment termed Homology Domain 1 (HD1), spanning roughly amino acids 259-314.9Unlike many classical leucine-rich NES motifs that interact with the exportin CRM1, the Msn2 NES is recognized by the exportin Msn5.9 Its activity is also regulated by PKA-mediated phosphorylation, but in a manner opposite to NLS regulation: phosphorylation at sites such as S288 and S304 within or near HD1 activates the NES, promoting Msn2's export from the nucleus.10 The conserved motif S$^{304}IS^{306}$HxxDFW within HD1 is particularly noteworthy.9 This dual regulatory mechanism—PKA-mediated inhibition of import via the NLS and PKA-mediated activation of export via the NES—provides a robust system for maintaining Msn2 in the cytoplasm under normal growth conditions. When stress signals lead to a decrease in PKA activity and subsequent dephosphorylation of these sites, nuclear import is favored and export is reduced, leading to rapid nuclear accumulation of Msn2.
Caption for the link above: The Nuclear Export Signal (NES) of Msn2 (minimal region ~237-327), containing Homology Domain 1 (HD1, residues 259-314, shown in darker teal), crucial for export from the nucleus, is highlighted in cyan/teal cartoon.3.5. Intrinsically Disordered Regions (IDRs): Flexible ModulatorsA striking feature of Msn2 is the prevalence of Intrinsically Disordered Regions (IDRs). A significant portion of the Msn2 sequence, particularly the large N-terminal region (upstream of the NES) and various linker segments, is predicted to lack a stable, well-defined three-dimensional structure.11 UniProt entry A2Q129, for example, annotates disordered regions at approximately residues 82-246, 411-437, and 592-634 (this last one overlapping with the NLS).13 These predictions are generally consistent with the lower pLDDT scores observed in these areas of the AlphaFold model (see Script 1).Far from being mere passive linkers, the IDRs of Msn2 are functionally critical. The N-terminal IDR harbors the transcriptional activation domains (TADs) necessary for Msn2 to recruit the transcriptional machinery and activate gene expression.8 Specific short motifs within this N-terminal IDR, such as "Motif A" (residues 1-50) and "Motif B" (residues 253-267), have been identified as important for transcriptional activity and interaction with components of the Mediator complex, like Gal11.11 More broadly, Msn2's IDRs are implicated in directing promoter selection and recruiting coactivators, employing what has been described as "interwoven sequence grammars" to encode these multiple functions.19 This suggests that different segments or physicochemical properties distributed across the IDRs contribute to distinct molecular interactions and regulatory outcomes.The inherent flexibility of IDRs allows them to adopt various conformations, facilitating interactions with a diverse range of binding partners. This dynamic nature is challenging to capture in a single static structural model. Therefore, when viewing the AlphaFold model, regions with low pLDDT scores, corresponding to predicted IDRs, should be interpreted not as poorly modeled structured domains but as representations of highly flexible and dynamic segments that are crucial for Msn2's regulatory capabilities.
Caption for the link above: Major predicted Intrinsically Disordered Regions (IDRs) of Msn2 are shown as a light salmon cartoon, with specific functional motifs A and B within the N-terminal IDR highlighted in orange. Note their typically lower pLDDT scores (see first script).4. Structural Basis of Msn2 Regulation: Focus on PhosphorylationPhosphorylation stands as the primary and most extensively studied post-translational modification regulating Msn2 activity, particularly its nucleocytoplasmic shuttling.1 Protein Kinase A (PKA) is the principal kinase responsible for phosphorylating Msn2 under non-stress conditions, leading to its cytoplasmic retention.1 Stress signals typically lead to a decrease in PKA activity (and/or activation of phosphatases), resulting in Msn2 dephosphorylation at key sites and its subsequent nuclear import.Several specific serine residues targeted by PKA (and in one case, Snf1/AMPK) have been identified, and their phosphorylation status directly impacts the functionality of the NLS and NES:
NES-related phosphorylation sites (within or near HD1, promoting export):
Serine 288 (S288): Located within a PKA consensus sequence (RRxS), phosphorylation of S288 is crucial for NES activity and thus for Msn2 export from the nucleus.10 Serine 304 (S304): This site is also important for NES function; its phosphorylation, which is responsive to glucose starvation, promotes export.10 Serines 306 and 308 in this vicinity are also implicated.10
NLS-related phosphorylation sites (inhibiting import):
Serine 582 (S582): A target for both PKA and the Snf1 kinase (yeast AMPK homolog), phosphorylation at S582 inhibits NLS function and nuclear import.10 Serine 620 (S620), Serine 625 (S625), and Serine 633 (S633): These are well-characterized PKA sites within the NLS region. Their phosphorylation blocks Msn2 import.10
DNA-Binding Domain (DBD)-proximal phosphorylation site:
Serine 686 (S686): This PKA consensus site is located near the zinc finger domains. Its phosphorylation may play a role in modulating DNA binding affinity or transcriptional activity once Msn2 is bound to DNA.10
The AlphaFold model allows for the visualization of these key serine residues in their 3D context. Their surface accessibility is a critical factor for interaction with kinases and phosphatases. Examining their locations can provide insights into how these modifications might induce conformational changes or alter protein-protein interactions that ultimately control Msn2's localization and activity. The clustering of multiple phosphorylation sites within the NLS and NES regions highlights how Msn2's activity can be fine-tuned by the integrated input from various signaling pathways, allowing for a graded response rather than a simple on/off switch. This multi-site phosphorylation represents a sophisticated regulatory code.It is also known that Msn2 becomes hyperphosphorylated upon stress, which might seem to contradict the dephosphorylation-driven nuclear import model.2 This stress-induced hyperphosphorylation likely occurs once Msn2 is in the nucleus and is thought to be related to its full transcriptional activation, possibly through interactions with the Mediator complex.11 For this molecular tour, the focus remains on the PKA sites directly controlling nucleocytoplasmic shuttling.
Caption for the link above: Key regulatory phosphorylation sites in Msn2 are shown as orange spheres. These sites, primarily targeted by PKA, control Msn2's nuclear localization and activity. NLS-related sites: S582, S620, S625, S633. NES-related sites: S288, S304. DBD-proximal site: S686.5. Putting It All Together: Msn2's Dynamic Structure and FunctionThe molecular tour of Msn2 reveals a protein exquisitely designed for its role as a central stress-responsive transcription factor. Its modular architecture, featuring distinct C2H2 zinc fingers for DNA binding, a regulatable Nuclear Localization Signal, a similarly controlled Nuclear Export Signal, and extensive N-terminal Intrinsically Disordered Regions housing transcriptional activation domains, allows for a multifaceted and finely tuned response to environmental cues.2The interplay between these structural elements is critical. The IDRs are not merely unstructured segments but functional hubs that provide conformational flexibility, mediate protein-protein interactions (e.g., with co-activators and the Mediator complex), and contribute to promoter selectivity.11 The zinc fingers provide the sequence specificity to target STREs in hundreds of gene promoters.1 The NLS and NES act as gatekeepers, controlling Msn2's access to these nuclear targets.7Phosphorylation serves as the key regulatory mechanism orchestrating these activities. The coordinated phosphorylation and dephosphorylation of multiple sites within the NLS and NES by kinases like PKA and opposing phosphatases allow the cell to rapidly control Msn2's subcellular localization in response to stress.10 This multi-site modification system enables a more nuanced, graded response to varying types and intensities of stress, rather than a simple binary switch. This complex regulation facilitates phenomena such as the "stochastic pulsing" of Msn2 into the nucleus, where Msn2 exhibits irregular, cell-autonomous oscillations between the nucleus and cytoplasm even under basal conditions, with stress modulating the frequency, duration, or amplitude of these pulses.4 Such dynamic behavior, likely amplified by the low copy number of Msn2 molecules and noise in signaling pathways, is facilitated by its flexible structural properties and intricate regulatory network.14The AlphaFold model provides a valuable static snapshot—a highly plausible 3D hypothesis—of this dynamic protein. The pLDDT scores inherently guide our interpretation, highlighting regions of stable fold versus those of inherent flexibility. It is crucial to remember that Msn2 in vivo exists as an ensemble of conformations, particularly its IDRs, and its function is intimately tied to these dynamics and its interactions with numerous cellular partners. Msn2 thus serves as an excellent paradigm for how transcription factors integrate cellular signals through a combination of structured domains, flexible regions, and precisely targeted post-translational modifications to orchestrate complex biological responses.6. Explore Msn2 Further with JSmolThis molecular tour has highlighted some of the key structural and functional features of the Msn2 protein. The provided JSmol interactive links allow for a dynamic exploration of these features. Readers are encouraged to use these scenes as a starting point for their own investigations. By using the JSmol console (often accessible via a right-click menu within the JSmol applet in Proteopedia, then selecting "Console"), you can enter your own JSmol commands to further manipulate the view, select different residues, change colors and representations, or measure distances.For comprehensive JSmol scripting guidance, the following resources are invaluable:
Jmol Interactive Scripting Documentation: https://chemapps.stolaf.edu/jmol/docs/ 22
Proteopedia's Help Pages on JSmol and Scene Authoring: Consult Proteopedia's internal help sections, such as those for "Jmol" or "Scene Authoring Tools," for tips on integrating JSmol within the Proteopedia environment.24
By combining the structural information presented here with the power of JSmol, a deeper understanding of how Msn2's architecture underpins its critical role in the yeast stress response can be achieved.
