The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate it by lowering its stability. We determined its structure in solution using state-of-the-art isotopic labeling techniques and NMR spectroscopy to complement its crystal structure. The structure was very similar to that in the crystal but far more mobile than expected. Importantly, we were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. We mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal/mol. We also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to the presence of alternative hydrogen-bond patterns in the protein interior. p53 appears to have evolved to be dynamic and unstable.

Vertebrate polyadenylation sites are identified by the AAUAAA signal and by GU‐rich sequences downstream of the cleavage site. These are recognized by a heterotrimeric protein complex (CstF) through its 64 kDa subunit (CstF‐64); the strength of this interaction affects the efficiency of poly(A) site utilization. We present the structure of the RNA‐binding domain of CstF‐64 containing an RNA recognition motif (RRM) augmented by N‐ and C‐terminal helices. The C‐terminal helix unfolds upon RNA binding and extends into the hinge domain where interactions with factors responsible for assembly of the polyadenylation complex occur. We propose that this conformational change initiates assembly. Consecutive Us are required for a strong CstF–GU interaction and we show how UU dinucleotides are recognized. Contacts outside the UU pocket fine tune the protein–RNA interaction and provide different affinities for distinct GU‐rich elements. The protein–RNA interface remains mobile, most likely a requirement to bind many GU‐rich sequences and yet discriminate against other RNAs. The structural distinction between sequences that form stable and unstable complexes provides an operational distinction between weakly and strongly processed poly(A) sites.

The tumor suppressor p53 consists of four 393-residue chains, each of which has two natively unfolded (N- and C-terminal) and two folded (core and tetramerization) domains. Their structural organization is poorly characterized as the protein tends to aggregate, has defied crystallization, and is at the limits of NMR studies. We first stabilized the protein by mutation to make it more suitable for extended study and then acquired NMR spectra on full-length protein and various combinations of shorter domain constructs. The NMR spectrum ( ¹⁵ N, ¹ H transverse relaxation optimized spectroscopy) of full-length p53 was close to that expected from the sum of the spectra of isolated individual domains. However, patterns of changes in chemical shifts revealed unexpected interactions between the core domains. We used the NMR data as constraints in docking algorithms and found a previously uncharacterized self-complementary surface for the association of core domains into dimers within the tetrameric complex. Binding to DNA requires about a 70° rotation to break those subunit interactions and form the known protein:protein interface in the p53–DNA complex. We verified the interactions by the effects of mutation on DNA binding. Spectroscopic, biophysical, and mutational data conspired to give a picture of the p53 tetramer as a dimer of loosely tethered core dimers of appropriate symmetry to be poised to bind target DNA.

The recognition of specific signals encoded within the 3′‐untranslated region of the newly transcribed mRNA triggers the assembly of a multiprotein machine that modifies its 3′‐end. Hrp1 recognises one of such signals, the so‐called polyadenylation enhancement element (PEE), promoting the recruitment of other polyadenylation factors in yeast. The molecular bases of this interaction are revealed here by the solution structure of a complex between Hrp1 and an oligonucleotide mimicking the PEE. Six consecutive bases (AUAUAU) are specifically recognised by two RNA‐binding domains arranged in tandem. Both protein and RNA undergo significant conformational changes upon complex formation with a concomitant large surface burial of RNA bases. Key aspects of RNA specificity can be explained by the presence of intermolecular aromatic–aromatic contacts and hydrogen bonds. Altogether, the Hrp1–PEE structure represents one of the first steps towards understanding of the assembly of the cleavage and polyadenylation machinery at the atomic level.

CCL20/MIP-3α is a β-chemokine expressed in the thymus, skin, and intestinal epithelial cells that exclusively binds and activates the CCR6 receptor in both mice and humans. The strict receptor binding specificity of CCL20 is exceptional; other chemokines and their receptors bind promiscuously with multiple partners. Toward determining the structural basis for the selective receptor specificity of CCL20, we have determined its three-dimensional structure by ¹H NMR spectroscopy. CCL20 exhibits the same monomeric structure previously described for other chemokines: a three-stranded β-sheet and an overlying α-helix. The CCL20 receptor selectivity could arise from the rigid conformation of the N-terminal DCCL motif as well as the groove between the N-loop and the β₂-β₃ hairpin, which is significantly narrower in CCL20 than in other chemokines. Similar structural features are seen in human β-defensin 2, a small nonchemokine polypeptide reported to selectively bind and activate CCR6, which stresses their importance for the specific binding of both CCL20 and β-defensin 2 to CCR6. CCL20's structure will be useful to design tools aimed to modulate its important biological functions.