Proteome-wide analysis of phospho-regulated PDZ domain interactions through phosphomimetic proteomic peptide phage display

Sundell, Gustav; Arnold, Roland; Ali, Muhammad; Orts, Julien; Guentert, P.; Celestine, N.; Ivarsson, Ylva

doi:10.1101/211250

Cited by 2 publications

(11 citation statements)

References 37 publications

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“…It is interesting to note that residues 741-744 in the b2 strand that flanks the peptide binding groove seem to respond differently to binding of the p-3 phosphorylated peptide and unphosphorylated peptide ligands. These results are in agreement with our mutational analysis of R762 to alanine, where the R762A mutant showed a decrease in affinity for the p-3 phosphorylated peptide compared to the unphosphorylated peptide 19 . This further indicates that our analysis can identify subtle differences important for PDZ affinity.…”

Section: The Sign Rather Than the Magnitude Of Nmr Chemical Shift Dissupporting

confidence: 92%

“…The two phosphorylated ligands are probes for the phosphorylation sites at p-3 and p-1, thus allowing the study of phosphorylation in a position-dependent context. The peptides were chosen as their position-dependent phosphorylation seemed to play a biological role as switches for different PDZ domains. , …”

Section: Resultsmentioning

confidence: 99%

“…In contrast, the directionality of the chemical shifts, which is rarely taken into consideration in this analysis, can play a vital role in distinguishing binding mechanisms, for example, when two slightly different ligands interact with the same receptor. The peptides MKRLTSTRL and MKRLTpSTRL, which differ at a single phosphorylation site, both bind to PDZ1 from Scribble, but the phosphorylated variant has a higher affinity (a K D of 18 μM vs a K D of 4 μM from ITC) . We reanalyzed HSQC experiments performed for free and bound Scribble PDZ1 with peptides phosphorylated at positions p-1 and p-3 as well as unphosphorylated peptides.…”

Section: Resultsmentioning

confidence: 99%

“…The Scribble PDZ1 domain has been shown to selectively bind to the C-terminal peptides of RPS6KA2 (MKRLTSTRL) 19 and colorectal mutant cancer protein (MCC) (RPHTNETSL), 20 and the interactions are tuned in a Ser phosphorylationdependent manner. 19 In this study, we investigate the molecular origin of these differences by analyzing chemical shift perturbations from heteronuclear single-quantum coherence spectra as well as slow millisecond dynamics probed by NMR transverse relaxation rates (R 2 ). The following peptides were used for analysis: unphosphorylated ligands MKRLTSTRL (RPS6KA2) and RPHTNETSL (MCC) and their MKRLTpS-TRL and RPHTNETpSL phosphorylated variants, respectively.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…We recently found that Scribble PDZ1 domain can interact with unphosphorylated and phosphorylated ligands representing the C-termini of different biological proteins. Depending on the phosphorylation site, phosphorylation may have a switching function, increasing the affinity for Scribble PDZ1 and decreasing the affinity for other PDZ domains such as Shank1 PDZ 19 . Although we probed the molecular mechanism of interaction through NMR structure determination, with hindsight we realized that, the sign shift analysis provides an alternative and feasible approach for gaining deeper understanding of the molecular details of the interactions.…”

Section: Introductionmentioning

confidence: 99%

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The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1

et al. 2017

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The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the N/H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

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Section: The Sign Rather Than the Magnitude Of Nmr Chemical Shift Dissupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1

et al. 2017

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The activation, operation and regulation of the mitotic kinase RSK1

Gógl¹

2018

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together by the adenine moiety of the bound cofactor. In contrast, the regulatory spine, or R-spine, is highly dynamic, and its assembly is required for catalytic activity (Kim et al., 2017). The R-spine includes a residue from the C-helix of the N-lobe, which serves as an allosteric hotspot for most protein kinases. This helix is also responsible for stabilizing a salt bridge between the bound nucleotide substrate and a Lys from β3. In inactive kinases the C-helix is often in an "out" or inactive conformation or disordered. (From now on, I will refer to the "nucleotide substrate" as a "cofactor" for simplification.) Embedded within the kinase fold is a functional intrinsically disordered region (IDR), the activation loop. The activation segment starts with the DFG motif and ends with the APE motif.These two flexible sequence motifs are conserved among kinases and have unique functions. The DFG motif lies between the two lobes, right below the C-helix and the Glycine-rich loop (G-loop).Embedded in the DFG motif is another R-spine residue, which couples directly with the C-Helix Rspine residue and the catalytic loop R-spine residue. Many kinases display two conformations: DFG-in and DFG-out (Hari et al., 2013). In the DFG-out conformation the R-spine is disassembled and in many cases the Phe side chain of this motif faces towards the G-loop instead of the C-helix.The DFG-out conformation is thus incompatible with ATP cofactor binding. Interplay between the DFG motif and the C-Helix in/out is highly regulated and defines the switch mechanism that leads to kinase activation. Both motifs are flexible and must be aligned.The APE motif precedes the F-helix in the C-lobe. Although it serves as a stable anchor for the flexible activation loop (AL), multiple crystal structures captured inter-molecularly bound APE motifs in kinase homodimers (Marcotte et al., 2017). In extreme cases, the APE motif can be disordered (Malakhova et al., 2008). These findings suggest that the APE motif is dynamically anchored to the C-lobe. The activation loop between the DFG and the APE motifs is usually 20-30 residues long and is often partially disordered in inactive structures. Although the activation loop is highly divergent, it usually contains at least one phosphorylation site, which orders and stabilizes the activation loop and is cooperatively coordinated by an Arg from the catalytic HRD motif (from the C-lobe), a basic residue in the activation loop, and a basic residue from the C-helix (from the Nlobe).Different kinase families developed orthogonal strategies to regulate the core kinase domain.This thesis focuses on RSK1, a tandem kinase, which includes an AGC-and a CaMK-type domains.Apparently, these two families are probably the most complicatedly regulated kinase families, which mainly organized around their C-terminal extensions.

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Proteome-wide analysis of phospho-regulated PDZ domain interactions through phosphomimetic proteomic peptide phage display

Cited by 2 publications

References 37 publications

The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1

The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1

The activation, operation and regulation of the mitotic kinase RSK1

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