Biochemistry Biochemistry最新文献

Mycobacterium tuberculosis (Mtb) is a leading cause of death, with an escalating global occurrence of drug-resistant infections that are partially attributed to cell wall mycolic acids derived from type II fatty acid biosynthesis (FAS-II). Here, the central acyl carrier protein, AcpM, contributes to the regulation of complex and specific protein–protein interactions (PPIs), though the orchestration of these events remain largely unresolved due to unique features of AcpM. Limitations include complexities in generating modified AcpM in a single state. Herein, we report a streamlined method to generate homogeneous samples of modified AcpM for applications in structure and functional studies. We apply these to generate solvatochromic labeled crypto-AcpM, where fluorescence response reports cargo sequestration and chain flipping upon interaction with four FAS-II enzymes. We find an increased fluorescence in a truncated form, AcpM80, indicating that the 35-residue C-terminus is involved in modulating the chemical environment surrounding the substrate and contributing to the regulation of PPIs. This study establishes an efficient chemo-enzymatic strategy to generate AcpM analogs for biophysical studies to aid in understanding the processes driving Mtb pathogenicity and drug resistance.

In the wake of the pandemic, peptidyl protease inhibitors with Pro-based rigid Leu mimetics at the P₂ position have emerged as potent drug candidates against the SARS-CoV-2 main protease. This success is intuitively attributed to the enhanced hydrophobic interactions and rigidity of Pro-based rigid Leu mimetics in the literature. However, the tertiary amide of proline P₂ derivatives, which hinders the formation of a critical hydrogen bond with the enzyme active site, and the constrained PP_II conformation, which contradicts the protease preferred β-strand conformation, represent two overlooked disadvantages associated with these inhibitors over traditional inhibitors and, theoretically, should adversely affect their potency. Interestingly, despite these major disadvantages, they maintain or display improved potency compared to traditional peptidyl protease inhibitors. In this study, we uncover a previously unnoticed preference for P₂ residues of the protease inhibitors to adopt the PP_II conformation, regardless of residue identity, in the main protease-bound form of key RNA viruses, deviating from the traditional β-strand conformation. We also demonstrate that Pro-based rigid Leu mimetics at P₂ enhance binding affinity by favoring the enzyme-preferred PP_II conformation and significantly reducing configurational entropy loss upon binding, comparable to that of a typical hydrogen bond. This work also highlights the importance of a multidisciplinary approach to enhance the understanding of structure–activity relationships beyond traditional medicinal chemistry intuition. We believe these findings provide new, deep insights and address a major knowledge gap in the area of peptidyl protease inhibitor design, identifying key drivers behind the success of Pro-based peptidyl protease inhibitors beyond mere rigidity and hydrophobicity.

Farnesyl pyrophosphate derivatives bearing an additional oxygen atom at position 5 proved to be very suitable for expanding the substrate promiscuity of sesquiterpene synthases (STSs) and the formation of new oxygenated terpenoids. Insertion of an oxygen atom in position 9, however, caused larger restraints that led to restricted acceptance by STSs. In order to reduce some of the proposed restrictions, two FPP-ether derivatives with altered substitution pattern around the terminal olefinic double bond were designed. These showed improved promiscuity toward different STSs. Four new cyclized terpenoids with an embedded ether group were isolated and characterized. In the case of two cyclic enol ethers, also the corresponding “hydrolysis” products, linear hydroxyaldehydes, were isolated. Interestingly, all cyclization products originate from an initial 1 → 12 cyclization unprecedented when native farnesyl pyrophosphate serves as a substrate. We found that the most suitable FPP derivative with an additional oxygen at position 9 does not carry any methyl group on the terminal alkene, which likely reduces steric congestion when the preferred conformation for cyclization is adopted in the active site.

The pathogen-associated C-glucosyltransferase IroB is involved in the biosynthesis of salmochelins, C-glucosylated derivatives of enterobactin (Ent), which is a triscatecholate siderophore of enteric bacteria including Salmonella enterica and Escherichia coli. Here, we reassess the ability of IroB to C-glucosylate non-native triscatecholate mimics of Ent, which may have utility in the design and development of siderophore-based therapeutics and diagnostics. We establish TRENCAM (TC) and MECAM (MC), synthetic Ent analogs with tris(2-aminoethyl)amine- or mesitylene-derived backbones replacing the trilactone core of Ent, respectively, and their monoglucosylated congeners as substrates of IroB. Time course analyses and steady-state kinetic studies, which were performed under conditions that provide enhanced activity relative to prior studies, inform the substrate selectivity and catalytic efficiencies of this enzyme. We extend these findings to the preparation of a siderophore-antibiotic conjugate composed of monoglucosylated TC and ampicillin (MGT-Amp). Examination of its antibacterial activity and receptor specificity demonstrates that MGT-Amp targets pathogenicity because it shows specificty for the pathogen-associated outer membrane receptor IroN. Overall, our findings extend the biochemical characterization of IroB and its substrate scope and illustrate the ability to leverage a bacterial C-glucosyltransferase for non-native chemoenzymatic transformations along with potential applications of salmochelin mimics.

The pathogen-associated C-glucosyltransferase IroB is involved in the biosynthesis of salmochelins, C-glucosylated derivatives of enterobactin (Ent), which is a triscatecholate siderophore of enteric bacteria including Salmonella enterica and Escherichia coli. Here, we reassess the ability of IroB to C-glucosylate non-native triscatecholate mimics of Ent, which may have utility in the design and development of siderophore-based therapeutics and diagnostics. We establish TRENCAM (TC) and MECAM (MC), synthetic Ent analogs with tris(2-aminoethyl)amine- or mesitylene-derived backbones replacing the trilactone core of Ent, respectively, and their monoglucosylated congeners as substrates of IroB. Time course analyses and steady-state kinetic studies, which were performed under conditions that provide enhanced activity relative to prior studies, inform the substrate selectivity and catalytic efficiencies of this enzyme. We extend these findings to the preparation of a siderophore–antibiotic conjugate composed of monoglucosylated TC and ampicillin (MGT-Amp). Examination of its antibacterial activity and receptor specificity demonstrates that MGT-Amp targets pathogenicity because it shows specificty for the pathogen-associated outer membrane receptor IroN. Overall, our findings extend the biochemical characterization of IroB and its substrate scope and illustrate the ability to leverage a bacterial C-glucosyltransferase for non-native chemoenzymatic transformations along with potential applications of salmochelin mimics.

The effects of guanidinium hydrochloride (GdmCl) on two intrinsically disordered proteins (IDPs) are investigated using simulations of the self-organized polymer-IDP (SOP-IDP) model. The impact of GdmCl is taken into account using the molecular transfer model (MTM). We show that due to the dramatic reduction in the stiffness of the highly charged Prothymosin-α (ProTα) with increasing concentration of GdmCl ([GdmCl]), the radius of gyration (R_g) decreases sharply until about 1.0 M. Above 1.0 M, ProTα expands, caused by the swelling effect of GdmCl. In contrast, R_g of α-Synuclein (αSyn) swells as continuously as [GdmCl] increases, with most of the expansion occurring at concentrations less than 0.2 M. Strikingly, the amplitude of the small-angle X-ray scattering (SAXS) profiles for ProTα increases until [GdmCl] ≈ 1.0 M and decreases beyond 1.0 M. The [GdmCl]-dependent SAXS profiles for αSyn, which has a pronounced bump at small wave vector (q ∼ 0.5 nm^–1) at low [GdmCl] (≤0.2 M), monotonically decrease at all values of [GdmCl]. The contrasting behavior predicted by the combination of MTM and SOP-IDP simulations may be qualitatively understood by modeling ProTα as a strongly charged polyelectrolyte with nearly uniform density of charges along the chain contour and αSyn as a nearly neutral polymer, except near the C-terminus, where the uncompensated negatively charged residues are located. The precise predictions for the SAXS profiles as a function of [GdmCl] can be readily tested.

The effects of guanidinium hydrochloride (GdmCl) on two intrinsically disordered proteins (IDPs) are investigated using simulations of the self-organized polymer-IDP (SOP-IDP) model. The impact of GdmCl is taken into account using the molecular transfer model (MTM). We show that due to the dramatic reduction in the stiffness of the highly charged Prothymosin-α (ProTα) with increasing concentration of GdmCl ([GdmCl]), the radius of gyration (R_g) decreases sharply until about 1.0 M. Above 1.0 M, ProTα expands, caused by the swelling effect of GdmCl. In contrast, R_g of α-Synuclein (αSyn) swells as continuously as [GdmCl] increases, with most of the expansion occurring at concentrations less than 0.2 M. Strikingly, the amplitude of the small-angle X-ray scattering (SAXS) profiles for ProTα increases until [GdmCl] ≈ 1.0 M and decreases beyond 1.0 M. The [GdmCl]-dependent SAXS profiles for αSyn, which has a pronounced bump at small wave vector (q ∼ 0.5 nm^-1) at low [GdmCl] (≤0.2 M), monotonically decrease at all values of [GdmCl]. The contrasting behavior predicted by the combination of MTM and SOP-IDP simulations may be qualitatively understood by modeling ProTα as a strongly charged polyelectrolyte with nearly uniform density of charges along the chain contour and αSyn as a nearly neutral polymer, except near the C-terminus, where the uncompensated negatively charged residues are located. The precise predictions for the SAXS profiles as a function of [GdmCl] can be readily tested.

Large Stokes shift red fluorescent proteins (LSS-RFPs) are of growing interest for multicolor bioimaging applications. However, their photochemical mechanisms are not fully understood. Here, we employed the QM(XDW-CASPT2//CASSCF)/MM method to investigate the excited-state proton transfer and photoisomerization processes of the LSS-RFP mKeima starting from its cis neutral isomer. Upon excitation to the bright S₁ state in the Franck–Condon region, mKeima relaxes to a metastable minimum-energy state. From this short-lived species, two competing deactivation pathways are available: the excited-state proton transfer in the S₁ state, and the S₁ decay via the S₁/S₀ conical intersection as a result of the cis–trans photoisomerization. In comparison, the former is a dominant excited-state relaxation pathway, leading to the cis anionic isomer of mKeima in the S₁ state. This anionic intermediate then undergoes cis–trans photoisomerization after overcoming a barrier of approximately 10 kcal/mol in the S₁ state, which is followed by an excited-state decay via the S₁/S₀ conical intersection region. The efficient nonadiabatic decay of the cis anionic isomer of mKeima in the S₁ state inhibits the radiative process, leading to a weak emission around 520 nm observed experimentally. These findings shed important mechanistic light on the experimental observations and provide valuable insights that could help in the design of LSS-RFPs with superior fluorescence properties.

Large Stokes shift red fluorescent proteins (LSS-RFPs) are of growing interest for multicolor bioimaging applications. However, their photochemical mechanisms are not fully understood. Here, we employed the QM(XDW-CASPT2//CASSCF)/MM method to investigate the excited-state proton transfer and photoisomerization processes of the LSS-RFP mKeima starting from its cis neutral isomer. Upon excitation to the bright S₁ state in the Franck-Condon region, mKeima relaxes to a metastable minimum-energy state. From this short-lived species, two competing deactivation pathways are available: the excited-state proton transfer in the S₁ state, and the S₁ decay via the S₁/S₀ conical intersection as a result of the cis-trans photoisomerization. In comparison, the former is a dominant excited-state relaxation pathway, leading to the cis anionic isomer of mKeima in the S₁ state. This anionic intermediate then undergoes cis-trans photoisomerization after overcoming a barrier of approximately 10 kcal/mol in the S₁ state, which is followed by an excited-state decay via the S₁/S₀ conical intersection region. The efficient nonadiabatic decay of the cis anionic isomer of mKeima in the S₁ state inhibits the radiative process, leading to a weak emission around 520 nm observed experimentally. These findings shed important mechanistic light on the experimental observations and provide valuable insights that could help in the design of LSS-RFPs with superior fluorescence properties.

The Ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a formidable barrier to viral replication by generating stress granules (SGs) in response to viral infections. Interestingly, viruses, including SARS-CoV-2, have evolved defensive mechanisms to hijack SG proteins like G3BP1 for the dissipation of SGs that lead to the evasion of the host's immune responses. Previous research has demonstrated that the interaction between the NTF2-like domain of G3BP1 (G3BP1_NTF-2) and the intrinsically disordered N-terminal domain (NTD-N_1-25) of the N-protein plays a crucial role in regulating viral replication and pathogenicity. Interestingly, the current study identified an additional upstream stretch of residues (128KDGIIWVATEG138) (N_128-138) within the N-terminal domain of the N-protein (NTD-N_41-174) that also forms molecular contacts with the G3BP1 protein, as revealed through in silico analysis, site-directed mutagenesis, and biochemical analysis. Remarkably, WIN-62577, and fluspirilene, the small molecules targeting the conserved peptide-binding pocket in G3BP1_NTF-2, not only disrupted the protein-protein interactions (PPIs) between NTD-N_41-174 and G3BP1_NTF-2 but also exhibited significant antiviral efficacy against SARS-CoV-2 replication with EC₅₀ values of ∼1.8 and ∼1.3 μM, respectively. The findings of this study, validated by biophysical thermodynamics and biochemical investigations, advance the potential of developing therapeutics targeting the SG host protein against SARS-CoV-2, which may also serve as a broad-spectrum antiviral target.