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Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a fragment binder to KRasG12D and used 1H 15N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

Research was conducted to study the relationship of S100B(betabeta) protein family with the p53 tumor suppressor gene and its role in PKC-dependent phosphorylation and tetramer formation. Recombinant S100B(betabeta) was prepared in Escherichia coli strain HMS174(DE3) and later transformed with an expression plasmid. Fluorescence spectra were gathered using a NSLM-Amino series 2 fluorescence spectrophotometer. Results suggested that the C-terminal regulatory domain of p53 and S100B(betabeta) may reveal clues on the involvement of S100B(betabeta) in cell-cycle processes.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of singleparticle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptorbinding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the diheptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile. [ABSTRACT FROM AUTHOR]

Vitamin A is a necessary nutrient for all mammals, and it is required for the transcription of many genes and vital for vision. While fasting, the vitamin A alcohol form (Retinol) from storage in the liver is mobilized and transported through the bloodstream while bound to retinol binding protein (RBP). Details of how exactly vitamin A is released from RBP and taken into the cells are still unclear. As part of the effort to elucidate the specifics of this process, single-particle cryo-electron microscopy structural studies of STRA6 (the RBP receptor 75-kDa transmembrane receptor protein) were recently reported by Chen et al. (Science, 10.1126/science.aad8266, 2016). Interestingly, STRA6 from zebrafish was shown to be a stable dimer and bound to calmodulin (CaM), forming a 180-kDa complex. The topology of the STRA6 complex includes 18 transmembrane helices (nine per protomer) and two long horizontal intramembrane helices interacting at the dimer core (Chen et al., in Science, 10.1126/science.aad8266, 2016). CaM was shown to interact with three regions of STRA6, termed CaMBP1, CaMBP2, and CaMBP3, with the most extensive interactions involving CaMBP2. To further our understanding of Ca2+-dependence of CaM-STRA6 complex formation, studies of the structure and dynamic properties of the CaMBP2–CaM complex were initiated. For this, the 1HN, 13C, and 15N backbone resonance assignments of the 148 amino acid Ca2+-bound calmodulin protein bound to the 27-residue CaMBP2 peptide derived from STRA6 were completed here using heteronuclear multidimensional NMR spectroscopy. [ABSTRACT FROM AUTHOR]

X-ray crystallography at 2.10 [Angstrom] (Rfree = 0.257), 1.98 [Angstrom] (Rfree = 0.281), and 1.90 [Angstrom] (Rfree = 0.228) resolution was utilized to determine the structures of three compounds (SBi132, SBi1279, and SBi523) bound to [Ca.sup.2+]-S100B. The findings for SBi132, SBi279, and SBi523 bound to proximal sites on [Ca.sup.2+]-S100B could be applied to design a new class of molecule(s) that interacts with one or more of these binding sites simultaneously, and obtain novel tight binding inhibitors specific for blocking protein-protein interactions involving S100B.

The binding of human S100A4 to [Ca.sup.2+] proteins that are involved in tumor invasion and metastasis and peptides derived from the C-terminus of myosin-IIA are examined to characterize the interaction between myosin IIA and S100A4. The results revealed that S100A4 promotes myosin-IIA filament disassembly by binding a single polypeptide chain of the coiled coil.

Tb(super 3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate affects the rate of Ca(super 2+) ion dissociation. Comparisons of wild-type and calcium binding mutants of S100B (E31A, E72A and E31A + E72A) provide additional information about the way in which residues are involved in the 'calcium switch' in the wild-type S100B protein.

The structural characterization of zinc binding to calcium-loaded S100B is examined using high-resolution nuclear magnetic resonance (NMR) techniques, including structural characterization of this complex in solution at atomic resolution. It is found that there are relative changes in the relative positioning of the two EF-hand calcium-binding domains and the respective helices comprising these EF-hands.

Nuclear magnetic resonance spectroscopy reveals that amino acid residues His-15, His-25, Cys-84, His-85, and His-90 coordinate the zinc binding to the S100 protein, S100B, a calcium binding protein. Data indicate that S100B undergoes changes in the secondary structure with zinc binding extending helix 4 by one full turn.

Calmodulin (CaM) and S100A1 fine-tune skeletal muscle Ca2+ release via opposite modulation of the ryanodine receptor type 1 (RyR1). Binding to and modulation of RyR1 by CaM and S100A1 occurs predominantly at the region ranging from amino acid residue 3614-3640 of RyR1 (here referred to as CaMBD2). Using synthetic peptides, it has been shown that CaM binds to two additional regions within the RyR1, specifically residues 1975-1999 and 4295-4325 (CaMBD1 and CaMBD3, respectively). Because S100A1 typically binds to similar motifs as CaM, we hypothesized that S100A1 could also bind to CaMBD1 and CaMBD3. Our goals were: (1) to establish whether S100A1 binds to synthetic peptides containing CaMBD1 and CaMBD3 using isothermal calorimetry (ITC), and (2) to identify whether S100A1 and CaM modulate RyR1 Ca2+ release activation via sites other than CaMBD2 in RyR1 in its native cellular context. We developed the mouse model (RyR1D-S100A1KO), which expresses point mutation RyR1-L3625D (RyR1D) that disrupts the modulation of RyR1 by CaM and S100A1 at CaMBD2 and also lacks S100A1 (S100A1KO). ITC assays revealed that S100A1 binds with different affinities to CaMBD1 and CaMBD3. Using high-speed Ca2+ imaging and a model for Ca2+ binding and transport, we show that the RyR1D-S100A1KO muscle fibers exhibit a modest but significant increase in myoplasmic Ca2+ transients and enhanced Ca2+ release flux following field stimulation when compared to fibers from RyR1D mice, which were used as controls to eliminate any effect of binding at CaMBD2, but with preserved S100A1 expression. Our results suggest that S100A1, similar to CaM, binds to CaMBD1 and CaMBD3 within the RyR1, but that CaMBD2 appears to be the primary site of RyR1 regulation by CaM and S100A1. [ABSTRACT FROM AUTHOR]