Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif-mediated interactions.

The interaction between the transcription factor p53 and the ubiquitin ligase MDM2 results in the degradation of p53 and is well-studied in cancer biology and drug development. Available sequence data suggest that both p53 and MDM2-family proteins are present across the animal kingdom. However, the interacting regions are missing in some animal groups, and it is not clear whether MDM2 interacts with, and regulates p53 in all species. We used phylogenetic analyses and biophysical measurements to examine the evolution of affinity between the interacting protein regions: a conserved 12-residue intrinsically disordered binding motif in the p53 transactivation domain (TAD) and the folded SWIB domain of MDM2. The affinity varied significantly across the animal kingdom. The p53TAD/MDM2 interaction among jawed vertebrates displayed high affinity, in particular for chicken and human proteins (KD around 0.1 µM). The affinity of the bay mussel p53TAD/MDM2 complex was lower (KD = 15 µM) and those from a placozoan, an arthropod, and a jawless vertebrate were very low or non-detectable (KD > 100 µM). Binding experiments with reconstructed ancestral p53TAD/MDM2 variants suggested that a micromolar affinity interaction was present in the ancestral bilaterian animal and was later enhanced in tetrapods while lost in other linages. The different evolutionary trajectories of p53TAD/MDM2 affinity during speciation demonstrate high plasticity of motif-mediated interactions and the potential for rapid adaptation of p53 regulation during times of change. Neutral drift in unconstrained disordered regions may underlie the plasticity and explain the observed low sequence conservation in TADs such as p53TAD.

Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain.

Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.

Harbouring group B streptococci in a neonatal intensive care unit led to an outbreak among preterm infants.

We report a nosocomial outbreak with group B streptococci (GBS) in a level two neonatal intensive care unit (NICU) at Sachs' Children and Youth Hospital, Stockholm, Sweden, in 2014. There were five very preterm infants with severe late-onset septicaemia, and 10 further infants were colonised. Pulsed-field gel electrophoresis and multilocus sequence typing genetic characterisation showed that one GBS strain was the cause: serotype Ia, sequence type 23, clonal complex 23. The NICU environment cultures revealed GBS reservoirs on surfaces near sick and colonised patients. We identified workflows and guidelines that could increase the risks of nosocomial infections. Conclusion: This nosocomial GBS outbreak among preterm infants demonstrates that GBS can be harboured in the NICU environment.

Binding Kinetics of the Intrinsically Disordered p53 Family Transactivation Domains and MDM2.

Because of their prominent roles in cell-cycle regulation and cancer, the interaction between MDM2 and the intrinsically disordered transactivation domain (TAD) of p53 is exceptionally well-studied. However, although there are numerous computational studies on the interaction mechanism, there is a paucity of experimental data regarding the kinetics and mechanism. We have used stopped flow fluorescence to investigate the binding reaction between MDM2 and TAD from p53 as well as from its paralogs p63 and p73, and in particular, focused on the salt dependence of the interaction. The observed kinetics are consistent with a two-state mechanism within the time frame of the stopped flow methodology; thus, any conformational changes including the previously identified MDM2 lid dynamics must occur on a time scale <5 ms at 10 °C. The association rate constants are similar for the three TADs, and differences in the dissociation rate constants determine the various affinities with MDM2. In contrast to previous studies, we found a relatively small ionic-strength dependence for all three interactions, highlighting the large variation in the role of electrostatics among binding reactions of intrinsically disordered proteins (IDPs). The basal association rate constants in the absence of electrostatic interactions were relatively high (≥2 × 106 M-1 s-1 at 10 °C), suggesting that a large number of initial contacts may lead to a productive complex. Our findings support an emerging picture of "conformational funneling" occurring in the initial stages of interactions involving IDPs and that these early binding events can rely on hydrophobic as well as charge-charge interactions.

Evolution of the p53-MDM2 pathway.

BACKGROUND: The p53 signalling pathway, which controls cell fate, has been extensively studied due to its prominent role in tumor development. The pathway includes the tumor supressor protein p53, its vertebrate paralogs p63 and p73, and their negative regulators MDM2 and MDM4. The p53/p63/p73-MDM system is ancient and can be traced in all extant animal phyla. Despite this, correct phylogenetic trees including both vertebrate and invertebrate species of the p53/p63/p73 and MDM families have not been published. RESULTS: Here, we have examined the evolution of the p53/p63/p73 protein family with particular focus on the p53/p63/p73 transactivation domain (TAD) and its co-evolution with the p53/p63/p73-binding domain (p53/p63/p73BD) of MDM2. We found that the TAD and p53/p63/p73BD share a strong evolutionary connection. If one of the domains of the protein is lost in a phylum, then it seems very likely to be followed by loss of function by the other domain as well, and due to the loss of function it is likely to eventually disappear. By focusing our phylogenetic analysis to p53/p63/p73 and MDM proteins from phyla that retain the interaction domains TAD and p53/p63/p73BD, we built phylogenetic trees of p53/p63/p73 and MDM based on both vertebrate and invertebrate species. The trees follow species evolution and contain a total number of 183 and 98 species for p53/p63/p73 and MDM, respectively. We also demonstrate that the p53/p63/p73 and MDM families result from whole genome duplications. CONCLUSIONS: The signaling pathway of the TAD and p53/p63/p73BD in p53/p63/p73 and MDM, respectively, dates back to early metazoan time and has since then tightly co-evolved, or disappeared in distinct lineages.

Emergence and evolution of an interaction between intrinsically disordered proteins.

Protein-protein interactions involving intrinsically disordered proteins are important for cellular function and common in all organisms. However, it is not clear how such interactions emerge and evolve on a molecular level. We performed phylogenetic reconstruction, resurrection and biophysical characterization of two interacting disordered protein domains, CID and NCBD. CID appeared after the divergence of protostomes and deuterostomes 450-600 million years ago, while NCBD was present in the protostome/deuterostome ancestor. The most ancient CID/NCBD formed a relatively weak complex (Kd∼5 µM). At the time of the first vertebrate-specific whole genome duplication, the affinity had increased (Kd∼200 nM) and was maintained in further speciation. Experiments together with molecular modeling using NMR chemical shifts suggest that new interactions involving intrinsically disordered proteins may evolve via a low-affinity complex which is optimized by modulating direct interactions as well as dynamics, while tolerating several potentially disruptive mutations.

Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.