Chemical Shift Perturbation Research Articles

Hyaluronan-arginine enhanced and dynamic interaction emerges from distinctive molecular signature due to electrostatics and side-chain specificity

Hyaluronan is a natural carbohydrate polymer with a negative charge that fosters gel-like conditions crucial for its cellular functions and industrial applications. As a recognized ligand for proteins, understanding their mutual interactions provides solid ground to tune hyaluronan's gel properties using biocompatible peptides. This work employs NMR and molecular dynamics simulations to identify molecular motifs relevant to hyaluronan–peptide interactions using arginine, lysine, and glycine oligopeptides. Arginine-rich peptides exhibit the strongest binding to hyaluronan according to chemical shift perturbation measurements, followed distantly by the similarly charged lysine. This difference highlights the significance of electrostatics and the peculiarities of the guanidinium side chain in arginine, capable of non-polar interactions that further stabilize the binding. Additional nuclear Overhauser effect measurements do not show stable interaction partners, precluding strong and well-defined complexes. Finally, molecular simulations support our findings and show an extended but significant interaction region, especially for arginine, responsible for the observed enhanced binding, which can also promote cross-linking of hyaluronan polymers. Our findings pave the way for optimizing biocompatible peptides to alter hyaluronan gels' properties efficiently and also explain why hyaluronan–protein interaction typically involves positively charged arginine-rich regions also capable of forming hydrogen bonds and non-polar interactions.

NMR characterization of the structure of the intrinsically disordered region of human origin recognition complex subunit 1, hORC1, and of its interaction with G-quadruplex DNAs

Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1413−511). Here, we investigate the structural characteristics of hORC1413−511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1413−511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1413−511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin.

Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics

In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13C−1 H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13C-T2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.

Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos.

Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. Our previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, we conducted NMR titration experiments using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. Our results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. We have also performed docking simulations based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, we discussed the various modes in the interactions of Drk with Sos/Dos.

Intracellular Fate of the Photosensitizer Chlorin e4 with Different Carriers and Induced Metabolic Changes Studied by 1H NMR Spectroscopy.

Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is affected by nanoparticles. In a systematic study, high-resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle-forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion-edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration-dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP, whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA, and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration-dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline the important protective role of the polymeric carriers following cell internalization. Moreover, to our knowledge, for the first time, the current study allowed us to trace intracellular PS localization on an atomic level by NMR methods.

Hexamethylene amiloride binds the SARS-CoV-2 envelope protein at the protein-lipid interface.

The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS-CoV-2 E.

Identifying structural and dynamic changes during the Biliverdin Reductase B catalytic cycle.

Biliverdin Reductase B (BLVRB) is an NADPH-dependent reductase that catalyzes the reduction of multiple substrates and is therefore considered a critical cellular redox regulator. In this study, we sought to address whether both structural and dynamics changes occur between different intermediates of the catalytic cycle and whether these were relegated to just the active site or the entirety of the enzyme. Through X-ray crystallography, we determined the apo BLVRB structure for the first time, revealing subtle global changes compared to the holo structure and identifying the loss of a critical hydrogen bond that "clamps" the R78-loop over the coenzyme. Amide and Cα chemical shift perturbations were used to identify environmental and secondary structural changes between intermediates, with more distant global changes observed upon coenzyme binding compared to substrate interactions. NMR relaxation rate measurements provided insights into the dynamic behavior of BLVRB during the catalytic cycle. Specifically, the inherently dynamic R78-loop that becomes ordered upon coenzyme binding persists through the catalytic cycle while similar regions experience dynamic exchange. However, the dynamic exchange processes were found to differ through the catalytic cycle with several groups of residues exhibiting similar dynamic responses. Finally, both local and distal structural and dynamic changes occur within BLVRB that are dependent solely on the oxidative state of the coenzyme. Thus, through a comprehensive analysis here, this study revealed structural and dynamic alterations in BLVRB through its catalytic cycle that are not simply relegated to the active site, but instead, are allosterically coupled throughout the enzyme.

Structural polymorphism of the PH domain in TFIIH.

The general transcription factor TFIIH is a multi-subunit complex involved in transcription, DNA repair, and cell cycle in eukaryotes. In the human p62 subunit and the budding yeast Saccharomyces cerevisiae Tfb1 subunit of TFIIH, the pleckstrin homology (PH) domain (hPH/scPH) recruits TFIIH to transcription-start and DNA-damage sites by interacting with an acidic intrinsically disordered region in transcription and repair factors. Whereas metazoan PH domains are highly conserved and adopt a similar structure, fungal PH domains are divergent and only the scPH structure is available. Here, we have determined the structure of the PH domain from Tfb1 of fission yeast Schizosaccharomyces pombe (spPH) by NMR. spPH holds an architecture, including the core and external backbone structures, that is closer to hPH than to scPH despite having higher amino acid sequence identity to scPH. In addition, the predicted target-binding site of spPH shares more amino acid similarity with scPH, but spPH contains several key residues identified in hPH as required for specific binding. Using chemical shift perturbation, we have identified binding modes of spPH to spTfa1, a homologue of hTFIIEα, and to spRhp41, a homologue of the repair factors hXPC and scRad4. Both spTfa1 and spRhp41 bind to a similar but distinct surface of spPH by modes that differ from those of target proteins binding to hPH and scPH, revealing that the PH domain of TFIIH interacts with its target proteins in a polymorphic manner in Metazoa, and budding and fission yeasts.

Eltrombopag directly activates BAK and induces apoptosis

Small molecule direct BAK activators can potentially be used for the development of anti-cancer drugs or as tools to study BAK activation. The thrombopoietin receptor agonist eltrombopag (Eltro) inhibits BAX activation and BAX-mediated apoptosis. Here we report that, in contrast to its function as a BAX inhibitor, Eltro directly binds BAK but induces its activation in vitro. Moreover, Eltro induces or sensitizes BAK-dependent cell death in mouse embryonic fibroblasts (MEFs) and Jurkat cells. Chemical shift perturbation analysis by NMR indicates that Eltro binds to the BAK α4/α6/α7 groove to initiate BAK activation. Further molecular docking by HADDOCK suggests that several BAK residues, including R156, F157, and H164, play an important role in the interaction with Eltro. The introduction of an R156E mutation in the BAK α4/α6/α7 groove not only decreases Eltro binding and Eltro-induced BAK activation in vitro but also diminishes Eltro-induced apoptosis. Thus, our data suggest that Eltro directly induces BAK activation and BAK-dependent apoptosis, providing a starting point for the future development of more potent and selective direct BAK activators.

Structural basis for plastic glycolipid recognition of the C-type lectin Mincle

Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor for cord factor, trehalose dimycolate (TDM), which serves as a potent component of adjuvants. The recognition of glycolipids by Mincle, especially their lipid parts, is poorly understood. Here, we performed nuclear magnetic resonance analysis, revealing that titration of trehalose harboring a linear short acyl chain showed a chemical shift perturbation of hydrophobic residues next to the Ca-binding site. Notably, there were split signalsfor Tyr201 upon complex formation, indicating two binding modes for the acyl chain. In addition, most Mincle residues close to the Ca-binding site showed no observable signals, suggesting their mobility on an ∼ ms scale even after complex formation. Mutagenesis study supported two putative lipid-bindingmodes for branched acyl-chain TDM binding. These results provide novel insights into the plastic-binding modes of Mincle toward a wide range of glycol- and glycerol-lipids, important for rational adjuvant development.

Investigation of the influence of pH on the properties and morphology of gelatin hydrogels

AbstractThe behavior of gelatin hydrogels is influenced by the charges located on the amino acid side chains throughout the gelatin molecules. The presence and distribution of ionisable side chains influences the surface activity of gelatin and ultimately determines the material properties. Herein, we report the influence of pH on mechanical properties as studied by texture analysis supported by data from polarimetry, zeta potential, pH titrations and NMR experiments. When adjusted to more extreme pH values (pH 2 and 12), softer gelatin blocks were observed. However, at pH values close to the isoelectric point (pH 5–10), the material is firmer. This behavior is related to the helical content. At pH 2 and pH 12 the surface of the gelatin carries a net charge, positive and negative, respectively, that inhibits the formation of tight helices and lowers the physical crosslink network density. Chemical shift perturbations were observed for the acidic amino acids glutamic and aspartic acid, under acidic pH, where their peaks shifted to higher ppm. Intense amide signals were observed at acidic pH but diminished with increasing pH. This was due to an increase in the rate of chemical exchange between the solvent and peptide amide protons as the pH increases.

Simultaneous inhibition of two neutrophil serine proteases by the S. aureus innate immune evasion protein EapH2

Extracellular adherence protein domain (EAP) proteins are high-affinity, selective inhibitors of neutrophil serine proteases (NSP), including cathepsin-G (CG) and neutrophil elastase (NE). Most Staphylococcus aureus isolates encode for two EAPs, EapH1 and EapH2, that contain a single functional domain and share 43% identity with one another. Although structure/function investigations from our group have shown that EapH1 uses a globally similar binding mode to inhibit CG and NE, NSP inhibition by EapH2 is incompletely understood due to a lack of NSP/EapH2 cocrystal structures. To address this limitation, we further studied NSP inhibition by EapH2 in comparison with EapH1. Like its effects on NE, we found that EapH2 is a reversible, time-dependent, and low nanomolar affinity inhibitor of CG. We characterized an EapH2 mutant which suggested that the CG binding mode of EapH2 is comparable to EapH1. To test this directly, we used NMR chemical shift perturbation to study EapH1 and EapH2 binding to CG and NE in solution. Although we found that overlapping regions of EapH1 and EapH2 were involved in CG binding, we found that altogether distinct regions of EapH1 and EapH2 experienced changes upon binding to NE. An important implication of this observation is that EapH2 might be capable of binding and inhibiting CG and NE simultaneously. We confirmed this unexpected feature by solving crystal structures of the CG/EapH2/NE complex and demonstrating their functional relevance through enzyme inhibition assays. Together, our work defines a new mechanism of simultaneous inhibition of two serine proteases by a single EAP protein.

Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.

Structural Mechanism of Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Assembly in Lipid Bilayers Revealed by Solid-State NMR.

Synaptic vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including synaptobrevin-2 (Syb-2), syntaxin-1 (Syx-1), and SNAP-25. However, it remains controversial whether the formation of thoroughly contacted α-helical bundle from the SNARE motifs to the end of the transmembrane domains (TMDs) is necessary for SNARE-mediated membrane fusion. In this study, we characterized the conformation of Syb-2 in different assembly states using a combination of dipolar- and scalar-based solid-state NMR experiments in lipid bilayers. Our spectral analysis revealed a highly dynamic nature of the Syb-2 TMD with considerable α-helical contents. Chemical shift perturbation and mutational analysis indicated that the coupling between Syb-2 and Syx-1 TMDs mediated by residue Gly-100 of Syb-2 together with high mobility of the C-terminal segment of Syb-2 TMD are required for inner membrane merger. Our results provide new insights into the role of the Syb-2 TMD in driving membrane fusion, which improves the current understanding of the structural mechanism of SNARE complex assembly. This study highlights the significance of membrane environments in elucidating the mechanism of membrane proteins.

Metal interactions of α-synuclein probed by NMR amide-proton exchange.

The aberrant aggregation of α-synuclein (αS), a disordered protein primarily expressed in neuronal cells, is strongly associated with the underlying mechanisms of Parkinson's disease. It is now established that αS has a weak affinity for metal ions and that these interactions alter its conformational properties by generally promoting self-assembly into amyloids. Here, we characterised the nature of the conformational changes associated with metal binding by αS using nuclear magnetic resonance (NMR) to measure the exchange of the backbone amide protons at a residue specific resolution. We complemented these experiments with 15N relaxation and chemical shift perturbations to obtain a comprehensive map of the interaction between αS and divalent (Ca2+, Cu2+, Mn2+, and Zn2+) and monovalent (Cu+) metal ions. The data identified specific effects that the individual cations exert on the conformational properties of αS. In particular, binding to calcium and zinc generated a reduction of the protection factors in the C-terminal region of the protein, whereas both Cu(II) and Cu(I) did not alter the amide proton exchange along the αS sequence. Changes in the R2/R1 ratios from 15N relaxation experiments were, however, detected as a result of the interaction between αS and Cu+ or Zn2+, indicating that binding to these metals induces conformational perturbations in distinctive regions of the protein. Collectively our data suggest that multiple mechanisms of enhanced αS aggregation are associated with the binding of the analysed metals.

Abstract A033: KRAS codon 12 oncogenic mutations modulate protein conformation within the Switch II/Helix3 pocket

Abstract Up to 30% of cancers harbor activating mutations in one of three RAS genes, HRAS, NRAS, or KRAS, that drive uncontrolled cellular proliferation, alter cellular metabolism, movement, differentiation, and promote cell survival and tumor immune evasion. The frequency of mutation varies across alleles and specific substitutions, with KRAS mutations occurring in roughly 81% of patients, and KRAS G12D, KRAS G12V, and KRAS G12C substitutions constituting 33%, 23%, and 12% of these cases, respectively. Cancer types exhibit characteristic preferences for RAS substitutions, the reason for which is still unknown. Oncogenic amino acid substitutions have been found to endow subtle differences in biochemical properties which cannot be explained by existing X-ray structural models. In addition, it is unclear whether these translate to distinctions in downstream signaling potential. We have initiated this study with the overall goal of understanding the effect of oncogenic mutations on the structure and dynamic properties of KRAS4b proteins in solution, and to identify any ensuing differences in effector binding behavior. Here, we present the data that identifies the structural differences between the common mutants KRAS G12D and KRAS G12C and the wildtype protein in the active conformation using solution-state NMR and Molecular Dynamics (MD) simulations, that could likely be coupled with the differing capacities to engage downstream effectors. Chemical shift perturbation (CSP) analysis demonstrates that oncogenic mutations in codon 12 of KRAS4b induce significant conformational differences when compared to the wildtype protein. These observations are localized to the Switch 2 region. We validate these observations in three-dimensional structures and reveal critical differences that are conferred by these oncogenic mutants. The solved three-dimensional solution structures of KRAS G12C/T35S/C118S-GppNHp and KRAS G12D/T35S/C118S-GppNHp reveal remarkable conformational differences within the Switch II regions, with the G12D-mutant protein adopting a relatively more flexible Switch II conformation than the G12C-mutant. Our MD simulations at a 2 µs timescale support these observations. Furthermore, we observed downstream signaling differences between KRAS G12C and KRAS G12D mutants in several cell line systems. If observed structural differences between KRAS mutants are found to directly translate to effector binding affinity, it would provide critical insight into why cancer types display preferences for RAS substitutions, and would inform RAS-targeted therapeutics. Citation Format: Brian Smith, Alok K. Sharma, Roger Ma, Megan Rigby, Vandana Kumari, Dominic Esposito, Dwight Nissley, Frank McCormick, Anna E. Maciag. KRAS codon 12 oncogenic mutations modulate protein conformation within the Switch II/Helix3 pocket [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A033.

19F: A small probe for a giant protein

Herein we describe a method for the efficient production (∼90% fluorination) of 5-F-Trp human H ferritin via the selective incorporation of 19F into the side chain of W93 using 5-fluoroindole as the fluorinated precursor of the amino acid. Human H ferritin is a nanocage composed of 24 identical subunits, each containing a single Trp belonging to a loop exposed on the external surface of the protein nanocage. This makes 5-F-Trp a potential probe for the study of intermolecular interactions in solution by exploiting its intrinsic fluorescence. More interestingly, albeit the large size of the cage (12 nm external diameter, ∼500 kDa molecular mass) we observe a broad but well defined NMR 19F resonance that can be used for the dual purpose of detecting solution intermolecular interactions via chemical shift perturbation mapping and monitoring the uptake of ferritin by cells treated with ferritin-based drug carriers, the latter being an application area of increasing importance.

Diverse Roles of ScSERF in Modifying the Fibril Growth of Amyloidogenic Proteins.

The aggregation of amyloidogenic proteins is often related to the occurrence of neurodegenerative diseases, including fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Recently, the SERF protein family has been reported to have a significant regulatory effect on amyloid formation, but it is still unclear about the detailed mechanisms of SERF acting on different amyloidogenic proteins. Herein, nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy were used to explore interactions of ScSERF with three amyloidogenic proteins FUS-LC, FUS-Core, and α-Synuclein. NMR chemical shift perturbations reveal them sharing similar interaction sites on the N-terminal region of ScSERF. However, the amyloid formation of α-Synuclein protein is accelerated by ScSERF, while ScSERF inhibits fibrosis of FUS-Core and FUS-LC proteins. Both the primary nucleation and the total amount of fibrils produced are detained. Our results suggest a diverse role of ScSERF in regulating the fibril growth of amyloidogenic proteins.

Abstract 3844: A biophysical investigation into the binding interactions of novel anti-cancer inhibitors of atypical protein kinase C (aPKCs)

Abstract The aim of this study is to address the challenge of the expression of recombinant atypical protein kinase C (aPKCs) in a bacterial system and understand the binding interactions of novel anti-cancer inhibitors of aPKCs. The Protein kinase Cs (PKCs) expression at a high rate is a key indicator of cancer cell proliferation and survival. In particular, the over-expressions of aPKCs were found to be directly associated with various cancer cells in benign and malignant stages. Several cancer growth blockers (inhibitors) are available for targeted therapy. However, these inhibitors had to pass through rigorous in silico screening, in vitro, and in vivo assays before FDA approval. During the characterization studies of the protein, insights into binding interactions between aPKCs and drug molecules are crucial. For successive studies such as for biophysical characterization, the recombinant bioactive full-length PKC- ι (an aPKC) expression in the bacterial system is still a challenge. In addition, the binding interaction studies of aPKCs with inhibitors in physiological conditions providing structural details are limited to some X-ray crystallography structures and computational studies. Here we addressed these two challenges and report a protocol detailing the bioactive full-length and catalytic domain of PKC- ι expression and purification from the bacterial system. The further characterization of the recombinant protein by the determination of melting temperature, functional activity, and circular dichroism reported here provides important information on the characteristics of the protein in its native state. Moreover, we show the chemical interactions of five novel anti-cancer inhibitors ICA-1S (Nucleosidic homolog) 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide), ICA-1T (Phosphorylated nucleosidic homolog of ICA-1S, [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate), ACPD (2-acetyl-1,3-cyclopentanedione), DNDA (3,4-diaminonaphthalene-2,7-disulfonic acid) and ζ(ZETA)-Stat (8-hydroxy-1,3,6- naphthalenetrisulfonic acid) to locate their regions of binding on the catalytic domain of the aPKCs (PKC- ι and PKC- ζ). We have utilized the chemical shift changes obtained from various multi-dimensional Nuclear Magnetic Resonance (NMR) experiments. Moreover, we have applied the isothermal calorimetry method to understand the biophysical properties of the aPKCs in presence of these inhibitors. The observed biophysical properties of the protein and chemical shift perturbations in the amide backbone of the catalytic domain upon binding shed light on the molecular recognition process, binding characteristics, and atomic-level structural details of the aPKCs. Citation Format: Radwan Ebna Noor, Tracess B. Smalley, Mildred Acevedo-Duncan. A biophysical investigation into the binding interactions of novel anti-cancer inhibitors of atypical protein kinase C (aPKCs). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3844.

Allosteric inactivation of an engineered optogenetic GTPase

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.