RESUMO
Protein post-translational modifications (PTMs) play an intricate role in a diverse range of cellular processes creating a complex PTM code that governs cell homeostasis. Understanding the molecular build-up and the critical factors regulating this PTM code is essential for targeted therapeutic design whereby PTM mis-regulation is prevalent. Here, we focus on Pin1, a peptidyl-prolyl cis-trans isomerase whose regulatory function is altered by a diverse range of PTMs. Through employing advanced mass spectrometry techniques in combination with fluorescence polarization and enzyme activity assays, we elucidate the impact of combinatorial phosphorylation on Pin1 function. Moreover, two phosphorylation sites were identified whereby Ser71 phosphorylation preceded Ser16 phosphorylation, leading to the deactivation of Pin1's prolyl isomerase activity before affecting substrate binding. Together, these findings shed light on the regulatory mechanisms underlying Pin1 function and emphasize the importance of understanding PTM landscapes in health and disease.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/química , Fosforilação , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.
Assuntos
Proteínas 14-3-3 , Proteínas Proto-Oncogênicas c-mdm2 , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismoRESUMO
Molecular glues are powerful tools for the control of protein-protein interactions. Yet, the mechanisms underlying multi-component protein complex formation remain poorly understood. Native mass spectrometry (MS) detects multiple protein species simultaneously, providing an entry to elucidate these mechanisms. Here, for the first time, covalent molecular glue stabilization was kinetically investigated by combining native MS with biophysical and structural techniques. This approach elucidated the stoichiometry of a multi-component protein-ligand complex, the assembly order, and the contributions of covalent versus non-covalent binding events that govern molecular glue activity. Aldehyde-based molecular glue activity is initially regulated by cooperative non-covalent binding, followed by slow covalent ligation, further enhancing stabilization. This study provides a framework to investigate the mechanisms of covalent small molecule ligation and informs (covalent) molecular glue development.
RESUMO
Cyanobacteria, also known as blue-green algae, are ubiquitous organisms on the planet. They contain tremendous protein machineries that are of interest to the biotechnology industry and beyond. Recently, the number of annotated cyanobacterial genomes has expanded, enabling structural studies on known gene-coded proteins to accelerate. This review focuses on the advances in mass spectrometry (MS) that have enabled structural proteomics studies to be performed on the proteins and protein complexes within cyanobacteria. The review also showcases examples whereby MS has revealed critical mechanistic information behind how these remarkable machines within cyanobacteria function.
Assuntos
Cianobactérias , Proteômica , Proteômica/métodos , Cianobactérias/metabolismo , Biotecnologia/métodos , Proteínas de Bactérias/metabolismo , Espectrometria de Massas/métodosRESUMO
Protein post-translational modifications (PTMs) enable cells to rapidly change in response to biological stimuli. With hundreds of different PTMs, understanding these control mechanisms is complex. To date, efforts have focused on investigating the effect of a single PTM on protein function. Yet, many proteins contain multiple PTMs. Moreover, one PTM can alter the prevalence of another, a phenomenon termed PTM crosstalk. Understanding PTM crosstalk is critical; however, its detection is challenging since PTMs occur substoichiometrically. Here, we develop an enrichment-free, label-free proteomics method that utilizes high-field asymmetric ion mobility spectrometry (FAIMS) to enhance the detection of PTM crosstalk. We show that by searching for multiple combinations of dynamic PTMs on peptide sequences, a 6-fold increase in candidate PTM crosstalk sites is identified compared with that of standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows. Additionally, by cycling through FAIMS compensation voltages within a single LC-FAIMS-MS/MS run, we show that our LC-FAIMS-MS/MS workflow can increase multi-PTM-containing peptide identifications without additional increases in run times. With 159 novel candidate crosstalk sites identified, we envisage LC-FAIMS-MS/MS to play an important role in expanding the repertoire of multi-PTM identifications. Moreover, it is only by detecting PTM crosstalk that we can "see" the full picture of how proteins are regulated.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Mobilidade Iônica/métodos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Blue-green algae, also known as cyanobacteria, contain some of the most efficient light-harvesting complexes known. These large, colourful complexes consist of phycobiliproteins which are extremely valuable in the cosmetics, food, nutraceutical and pharmaceutical industries. Additionally, the colourful and fluorescent properties of phycobiliproteins can be modulated by metal ions, making them highly attractive as heavy metal sensors and heavy metal scavengers. Although the overall quenching ability metal ions have on phycobiliproteins is known, the mechanism of heavy metal binding to phycobiliproteins is not fully understood, limiting their widespread quantitative applications. Here, we show using high-resolution native mass spectrometry that phycobiliprotein complexes bind metal ions in different manners. Through monitoring the binding equilibria and metal-binding stoichiometry, we show in particular copper and silver to have drastic, yet different effects on phycobiliprotein structure, both copper and silver modulate the overall complex properties. Together, the data reveals the mechanisms by which metal ions can modulate phycobiliprotein properties which can be used as a basis for the future design of metal-related phycobiliprotein applications.
Assuntos
Cianobactérias , Ficobiliproteínas , Cobre/metabolismo , Cianobactérias/metabolismo , Ficobiliproteínas/química , Ficobiliproteínas/metabolismo , Prata/metabolismoRESUMO
Cyanobacteria have evolved over billions of years to adapt and survive in diverse climates. Environmentally, this presents a huge challenge because cyanobacteria can now rapidly form algae blooms that are detrimental to aquatic life. In addition, many cyanobacteria produce toxins, making them hazardous to animals and humans that they encounter. Rapid identification of cyanobacteria is essential to monitor and prevent toxic algae blooms. Here, we show for the first time how native mass spectrometry can quickly and precisely identify cyanobacteria from diverse aquatic environments. By monitoring phycobiliproteins, abundant protein complexes within cyanobacteria, simple, easy-to-understand mass spectral "fingerprints" were created that were unique to each species. Moreover, our method is 10-fold more sensitive than the current MALDI-TOF mass spectrometric methods, meaning that cyanobacteria can be monitored using this technology prior to bloom formation. Together, the data show great promise for the simultaneous detection and identification of co-existing cyanobacteria in situ.
Assuntos
Cianobactérias , Toxinas Biológicas , Animais , Eutrofização , Humanos , Espectrometria de MassasRESUMO
Protein-protein interactions (PPIs) are key therapeutic targets. Most PPI-targeting drugs in the clinic inhibit these important interactions; however, stabilising PPIs is an attractive alternative in cases where a PPI is disrupted in a disease state. The discovery of novel PPI stabilisers has been hindered due to the lack of tools available to monitor PPI stabilisation. Moreover, for PPI stabilisation to be detected, both the stoichiometry of binding and the shift this has on the binding equilibria need to be monitored simultaneously. Here, we show the power of native mass spectrometry (MS) in the rapid search for PPI stabilisers. To demonstrate its capability, we focussed on three PPIs between the eukaryotic regulatory protein 14-3-3σ and its binding partners estrogen receptor ERα, the tumour suppressor p53, and the kinase LRRK2, whose interactions upon the addition of a small molecule, fusicoccin A, are differentially stabilised. Within a single measurement the stoichiometry and binding equilibria between 14-3-3 and each of its binding partners was evident. Upon addition of the fusicoccin A stabiliser, a dramatic shift in binding equilibria was observed with the 14-3-3:ERα complex compared with the 14-3-3:p53 and 14-3-3:LRRK2 complexes. Our results highlight how native MS can not only distinguish the ability of stabilisers to modulate PPIs, but also give important insights into the dynamics of ternary complex formation. Finally, we show how native MS can be used as a screening tool to search for PPI stabilisers, highlighting its potential role as a primary screening technology in the hunt for novel therapeutic PPI stabilisers.
RESUMO
O-GlcNAcylation is an abundant post-translational modification in the nervous system, linked to both neurodevelopmental and neurodegenerative disease. However, the mechanistic links between these phenotypes and site-specific O-GlcNAcylation remain largely unexplored. Here, we show that Ser517 O-GlcNAcylation of the microtubule-binding protein Collapsin Response Mediator Protein-2 (CRMP2) increases with age. By generating and characterizing a Crmp2S517A knock-in mouse model, we demonstrate that loss of O-GlcNAcylation leads to a small decrease in body weight and mild memory impairment, suggesting that Ser517 O-GlcNAcylation has a small but detectable impact on mouse physiology and cognitive function.
Assuntos
Acetilglucosamina/metabolismo , Cognição , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Memória de Curto Prazo , Proteínas do Tecido Nervoso/metabolismo , Acetilglucosamina/análise , Envelhecimento , Sequência de Aminoácidos , Animais , Linhagem Celular , Comportamento Exploratório , Feminino , Técnicas de Introdução de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Mutação Puntual , Processamento de Proteína Pós-TraducionalRESUMO
The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting ß-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Cristalografia por Raios X , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Periplasma/metabolismo , Ligação Proteica , Conformação Proteica em Folha betaRESUMO
Mass spectrometry is frequently used to determine protein complex topology. By combining in-solution and gas-phase dissociation measurements, information can be indirectly inferred about the original composition of the protein complex. Although the mechanisms behind gas-phase complex dissociation are becoming more established, protein complex dissociation is not always predictable. Here, we looked into the effect of the protein subunits pI on complex dissociation. We chose two structurally similar, hexameric protein complexes that consist of a ring of alternating alpha and beta subunits. For one complex, allophycocyanin, the alpha and beta subunits are structurally similar, almost identical in mass, but have distinct pIs. In contrast, the other complex, phycoerythrin, is structural similar to allophycocyanin, yet the subunits have identical pIs. As predicted based on the structural arrangement, dissociation of phycoerythrin resulted in the observation of both the alpha and beta monomeric subunits in the mass spectrometer. However, for allophycocyanin, the results differed dramatically, with only the alpha monomeric subunit being detected upon gas-phase dissociation. Together, the results highlighted the importance of considering the isoelectric points of individual subunits within a protein complex when using tandem mass spectrometry data to elucidate protein complex topology.
Assuntos
Proteínas de Bactérias/química , Ficobiliproteínas/química , Proteínas de Plantas/química , Porphyridium/química , Spirulina/química , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/químicaRESUMO
A wide variety of protein post-translational modifications (PTMs) decorate cellular proteins, regulating their structure, interactions and ultimately their function. The density of co-occurring PTMs on proteins can be very high, where multiple PTMs can positively or negatively influence each other's actions, termed PTM crosstalk. In this review, we highlight recent progress in the area of PTM crosstalk, whereby we focus on crosstalk between protein phosphorylation and O-GlcNAcylation. These two PTMs largely target identical (i.e., Ser and Thr) amino acids in proteins. Phosphorylation/O-GlcNAcylation crosstalk comes in many flavors, for instance by competition for the same site/residue (reciprocal crosstalk), as well as by modifications influencing each other in proximity or even distal on the protein sequence. PTM crosstalk is observed on the writers of these modifications (i.e., kinases and O-GlcNAc transferase), on the erasers (i.e., phosphatases and O-GlcNAcase), and on the readers and the substrates. We describe examples of all these different flavors of crosstalk, and additionally the methods that are emerging to better investigate in particular phosphorylation/O-GlcNAcylation crosstalk.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Animais , Glicosilação , Humanos , Fosforilação , Transdução de SinaisRESUMO
Phycoerythrin is the major light-harvesting pigment protein in red algae and is nowadays widely used as a fluorescent probe in biotechnological applications such as flow cytometry and immunofluorescence microscopy. In addition, it has had substantial economic impact due to its potential as a natural food colorant. However, knowledge on the precise molecular composition of phycoerythrin is limited. Here, we use a combination of high-resolution native mass spectrometry (MS) and fluorescence spectroscopy to characterize the assembly properties of the B-phycoerythrin protein complex from Porphyridium cruentum. Our data highlight the stabilizing role of the γ subunit in the intact B-phycoerythrin protein complex. In addition, by native MS we monitor B-phycoerythrin (dis)assembly intermediates, providing insight into which species contribute to B-phycoerythrins color and the factors that give B-phycoerythrin its highly fluorescent properties. Together, the data provide significant insights into the structural properties of B-phycoerythrin which is beneficial for its use within the biotechnology industry.
Assuntos
Corantes/química , Ficoeritrina/química , Pigmentos Biológicos/química , Estrutura Quaternária de Proteína , Cor , Corantes/metabolismo , Fluorescência , Espectrometria de Massas , Modelos Moleculares , Ficoeritrina/metabolismo , Pigmentos Biológicos/metabolismo , Porphyridium/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Espectrometria de FluorescênciaRESUMO
For many proteins, phosphorylation regulates their interaction with other biomolecules. Herein, we describe an unexpected phenomenon whereby phosphate groups are transferred non-enzymatically from one interaction partner to the other within a binding interface upon activation in the gas phase. Providing that a high affinity exists between the donor and acceptor sites, this phosphate transfer is very efficient and the phosphate groups only ligate to sites in proximity to the binding region. Consequently, such phosphate-transfer reactions may define with high precision the binding site between a phosphoprotein and its binding partner, as well as reveal that the binding site in this system is retained in the phase transfer from solution to the gas phase.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosfatos/metabolismo , Mapas de Interação de Proteínas , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/química , Humanos , Modelos Moleculares , Peptidilprolil Isomerase de Interação com NIMA/química , Fosfatos/química , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Proteins can be modified by multiple posttranslational modifications (PTMs), creating a PTM code that controls the function of proteins in space and time. Unraveling this complex PTM code is one of the great challenges in molecular biology. Here, using mass spectrometry-based assays, we focus on the most common PTMs-phosphorylation and O-GlcNAcylation-and investigate how they affect each other. We demonstrate two generic crosstalk mechanisms. First, we define a frequently occurring, very specific and stringent phosphorylation/O-GlcNAcylation interplay motif, (pSp/T)P(V/A/T)(gS/gT), whereby phosphorylation strongly inhibits O-GlcNAcylation. Strikingly, this stringent motif is substantially enriched in the human (phospho)proteome, allowing us to predict hundreds of putative O-GlcNAc transferase (OGT) substrates. A set of these we investigate further and show them to be decent substrates of OGT, exhibiting a negative feedback loop when phosphorylated at the P-3 site. Second, we demonstrate that reciprocal crosstalk does not occur at PX(S/T)P sites, i.e., at sites phosphorylated by proline-directed kinases, which represent 40% of all sites in the vertebrate phosphoproteomes.
Assuntos
Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Acetilglucosamina/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Glicosilação , Humanos , Espectrometria de Massas/métodos , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/fisiologia , Prolina , Proteínas/metabolismo , Proteólise , Serina , Transdução de Sinais , TreoninaRESUMO
O-GlcNAcylation is one of the most abundant metazoan nuclear-cytoplasmic post-translational modifications. Proteins modified by O-GlcNAc play key cellular roles in signaling, transcription, metabolism, and cell division. Mechanistic studies on protein O-GlcNAcylation are hampered by the lack of methods that can simultaneously quantify O-GlcNAcylation, determine its stoichiometry, and monitor O-GlcNAcylation kinetics. Here, we demonstrate that high-resolution native mass spectrometry can be employed to monitor the small mass shifts induced by modification by O-GlcNAc on two known protein substrates, CK2α and TAB1, without the need for radioactive labeling or chemoenzymatic tagging using large mass tags. Limited proteolysis enabled further localization of the O-GlcNAc sites. In peptide-centric MS analysis, the O-GlcNAc moiety is known to be easily lost. In contrast, we demonstrate that the O-GlcNAc is retained under native MS conditions, enabling precise quantitative analysis of stoichiometry and O-GlcNAcylation kinetics. Together, the data highlight that high resolution native MS may provide an alternative tool to monitor kinetics on one of the most labile of protein post-translational modifications, in an efficient, reliable, and quantitative manner.
Assuntos
Acetilglucosamina/química , Espectrometria de Massas , Proteínas/metabolismo , Acilação , Glicosilação , Cinética , Processamento de Proteína Pós-Traducional , Proteínas/químicaRESUMO
Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as "native MS," has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by "native MS," when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstract á .
Assuntos
Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , DNA/química , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização ProteicaRESUMO
Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as "native MS," has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by "native MS," when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions.
RESUMO
Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking-MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry-MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation.