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The CASP14 experiment demonstrated the extraordinary structure modeling capabilities of artificial intelligence (AI) methods. That result has ignited a fierce debate about what these methods are actually doing. One of the criticisms has been that the AI does not have any sense of the underlying physics but is merely performing pattern recognition. Here, we address that issue by analyzing the extent to which the methods identify rare structural motifs. The rationale underlying the approach is that a pattern recognition machine tends to choose the more frequently occurring motifs, whereas some sense of subtle energetic factors is required to choose infrequently occurring ones. To reduce the possibility of bias from related experimental structures and to minimize the effect of experimental errors, we examined only CASP14 target protein crystal structures determined to a resolution limit better than 2 Å, which lacked significant amino acid sequence homology to proteins of known structure. In those experimental structures and in the corresponding models, we track cis peptides, π- helices, 3 10- helices, and other small 3D motifs that occur in the PDB database at a frequency of lower than 1% of total amino acid residues. The best-performing AI method, AlphaFold2, captured these uncommon structural elements exquisitely well. All discrepancies appeared to be a con- sequence of crystal environment effects. We propose that the neural network learned a protein structure potential of mean force, enabling it to correctly identify situations where unusual structural features represent the lowest local free energy because of subtle influences from the atomic environment. [ABSTRACT FROM AUTHOR]

Transcription factor p63, a p53 family member, plays a role in epithelial cell development, cell cycle arrest, apoptosis, and tumorigenesis. Point mutations, primarily in the DNA binding domain (p63DBD). lead to malformation syndromes. To gain insight into differences between p63 and p53 and the impact of mutations on the structure, we have determined two crystal structures of p63DBD in complex with A/T-rich response elements. One complex contains a 10-bp DNA half-site response element (5'AAACATGT- TT3') and the other contains a 22-bp DNA full response element with a 2-bp spacer between two half-sites (5'AAACATGTTTTAAAA- CATGTTT3'). In both structures, each half-site binds a p63DBD dimer. The two p63DBD dimers do not interact in the presence of the DNA spacer, whereas they interact with one another in the p63DBD/10-bp complex where the DNA simulates a full response element by packing end-to-end. A unique dimer-dimer interaction involves a variable loop region, which differs in length and se- quence from the counterpart loop of p53DBD. The DNA trajectories in both structures assume superhelical conformations. Surface plasmon resonance studies of p63DBD/DNA binding yielded Kd = 11.7 μM for a continuous full response element, whereas binding was undetectable with the 22-bp DNA, suggesting an important contribution of a p63DBD interdimer interface to binding and establishing that p63DBD affinity to the response element is approximately 1,000-fold lower than that of p53DBD. Analyses of the structural consequences of p63DBD mutations that cause develop- mental defects show that, although some mutations affect DNA binding directly, the majority affects protein stability. [ABSTRACT FROM AUTHOR]

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg2+-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg2+ and PEP, followed by oxalate, an El inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg2+ and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189∼P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an α-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189∼P and His-15 are appropriately stationed for an in-line phosphotransfer reaction. [ABSTRACT FROM AUTHOR]