NMR-Guided Directed Evolution

Directed evolution can rapidly achieve dramatic improvements in the properties of a protein or bestow entirely new functions on it. We have discovered a strong correlation between the probability of finding a productive mutation at a particular position of a protein and a chemical shift perturbation in Nuclear Magnetic Resonance spectra upon addition of an inhibitor for the chemical reaction it promotes. In a proof-of-concept study we converted myoglobin, a non-enzymatic protein, into the most active Kemp eliminase reported to date using only three mutations. The observed levels of catalytic efficiency are on par with the levels shown by natural enzymes. This simple approach, that requires no a priori structural or bioinformatic knowledge, is widely applicable and will unleash the full potential of directed evolution.

Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413–511 of human ORC subunit 1, hORC1413–511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413–511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413–511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413–511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413–511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

Molecular mechanism of negative cooperativity of ferredoxin-NADP+ reductase by ferredoxin and NADP(H): involvement of a salt bridge between Asp60 of ferredoxin and Lys33 of FNR

ABSTRACT Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH at the end of the photosynthetic electron transfer chain. We previously showed that the interaction between FNR and Fd was weakened by the allosteric binding of NADP(H) on FNR, which was considered as a part of negative cooperativity. In this study, we investigated the molecular mechanism of this phenomenon using maize FNR and Fd, as the three-dimensional structure of this Fd:FNR complex is available. NMR chemical shift perturbation analysis identified a site (Asp60) on Fd molecule which was selectively affected by NADP(H) binding on FNR. Asp60 of Fd forms a salt bridge with Lys33 of FNR in the complex. Site-specific mutants of FdD60 and FNRK33 suppressed the negative cooperativity (downregulation of the interaction between FNR and Fd by NADPH), indicating that a salt bridge between FdD60 and FNRK33 is involved in this negative cooperativity.

Rational Design of 2,4-Disubstituted Quinazoline Small Molecules to Inhibit the Inflammatory Cytokine Oncostatin M

Inflammation is one of the body's most important natural defense mechanisms involved in wound healing. It is usually triggered by a harmful event, such as physical trauma or exposure to external stimuli including bacteria, fungi, viruses, harmful chemicals, or environmental particulates. The inflammatory process brings blood containing inflammatory mediators consisting of leukocytes, hormones, and cytokines to the site of trauma to begin healing. However, the lack of a proper inflammatory response or an overactive response can lead to further progressive tissue damage resulting in chronic inflammatory conditions or death. The cytokine oncostatin M (OSM) is of particular interest due to the pivotal role it plays in chronic inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, and various forms of cancer. These diseases have a detrimental impact on a person’s quality of life and life expectancy, as well as the economy and health care system. There is currently no clinically approved treatment targeting OSM. Thus, we propose the development of a small molecule inhibitor (SMI) targeting OSM. Using the known crystal structure of OSM combined with computational methods, a sample of 10,000 randomly selected molecules from online databases were docked in the OSM binding site 3, the site presumably responsible for binding to its receptor. The most energetically favorable binding poses were used to create a weighted density map (WDM) that shows the probability of aromatic carbons, hydrogen bond acceptors, and hydrogen bond donors to bind to OSM at particular locations in site 3. A 2,4-disubstituted quinazoline SMI was rationally designed that constructively overlaid with the WDM and was predicted to bind with high affinity based on computational docking studies. The SMI and analogs thereof, termed the SMI-27 series, were synthesized using a 4-step reaction sequence to create a small library to be tested against OSM. In order to evaluate the ability of the SMIs to inhibit OSM activity and to determine cytokine binding specificity, enzyme-linked immunosorbent assays (ELISAs) and western blot assays were performed. Fluorescence quenching experiments were used to determine the binding affinity of SMI analogs toward OSM. Finally, chemical shift perturbation NMR experiments were used to identify the important amino acids required for binding of the SMI to OSM. All of the SMI-27 analogs tested by ELISA inhibited OSM induced pSTAT3 expression below the level of the control. Additionally, SMIs 27B3 and 27B5 showed specific binding to OSM, and not to leukemia inhibitory factor (LIF) or interleukin-6 (IL-6), structurally related cytokines. The fluorescence quenching assays indicate that all SMIs exhibited direct binding to OSM, with 27B12 having a Kd of 5.1 ± 2.7 uM. Finally, the chemical shift perturbation assay identified several amino acids that appear to be involved in SMI binding. Importantly, three of these, tentatively assigned as Arg91, Leu92, and Gly166, are all located in OSM site 3. These experiments support our hypothesis that an SMI can be used to inhibit OSM activity and lay a solid foundation for the development of an SMI drug candidate that would provide a significant advancement in clinical treatments of OSM-related diseases.

Mechanistic Studies of the Stabilization of Insulin Helical Structure by Coomassie Brilliant Blue

Human insulin (HI) is an essential protein hormone and its biological activity mostly depends on folded and active conformation in the monomeric state. The present investigation established that Coomassie Brilliant Blue G-250 (CBBG), a small multicyclic hydroxyl compound can reversibly bind to the hormonal protein dimer and maintained most of α-helical folds crucial for biological function of the enzyme. The solution-state 1D NMR and isothermal calorimetric analysis showed a sub-micromolar binding affinity of the molecule to HI. 2D NOESY NMR established that the HI dimer undergoes residue level local conformational change upon binding to CBBG. The chemical shift perturbation and the NOE parameters of active protons of amino acid residues throughout the polypeptides further suggested that CBBG upon binding the protein stabilize α-helixes of both the A and B subunits of the hormonal protein. The changes in Gibb’s free energy (∆G) of the binding was of ~−11.1 kcal/mol and suggested a thermodynamically favourable process. The changes in enthalpy (∆H) and entropy term (T∆S) were −57.2 kcal/mol and −46.1 kcal/mol, respectively. The negative changes in entropy and the NOE transfer effectiveness of several residues in the presence of CBBG molecules indicated that the binding was an enthalpy driven favourable equilibrium process. The NMR-based atomic resolution data and molecular docking studies confirmed that the CBBG binds to HI at the dimeric stage and prevents the availability of the crucial residue segments that partake directly in further oligomerization and subsequent fibrillation. Extended computational analysis based on chemical shift perturbation of protons of active residues further established receptor-ligand based pharmacophore model comprised of 5 hydrophobic and a hydrogen bond acceptor features that can anchor the residues at the A and B chains of HI and inhibit the partial unfolding and hydrophobic collapse to nucleate the fibrillation. Taken together, the results demonstrated that CBBG and their close analogues might be useful to develop a formulation that will maintain the active and functional form of the hormonal protein for a significantly longer time.TOC

Structural and functional analyses of the N-terminal domain of the A subunit of aBacillus megateriumspore germinant receptor

Germination ofBacillusspores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore’s inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of theBacillus megateriumGerK3GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substrate-binding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination.

Study of CH/π Interactions in the Molecular Recognition between Acetyl Galactopyranoside and 6-substituted 2-Methoxypyridines and 2(1H)-Pyridones

A series of 6-substituted 2-methoxypyridine and 2(1<em>H</em>)-pyridones was designed and synthesized for its evaluation in the molecular recognition of acetyl 2,3,4,6-tetra-<em>O</em>-methyl-b-D-galactopyranoside substrate. ¹H-NMR titration (affinity constant <em>K</em>_a determination) and chemical shift perturbation experiments were performed to evaluate the capacity of these receptors to form CH/π interactions with the substrate. The addition of 2-methoxypyridines to the substrate effected up-field shift for the H³, H⁴ and H⁵ proton signals and down-field shift for the H² proton signal of galactopyranoside substrate. The determined affinity constant <em>K</em>_a values for the association between 2(1<em>H</em>)-pyridones and galactopyranoside showed that molecular recognition was weak. These results have demonstrated the existence of weak CH/π interactions and have reflected their weak intermolecular nature. Finally DFT calculations were performed to illustrate the geometry of the molecular recognition between 2(1<em>H</em>)-pyridones and galactopyranoside.