Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids

Enzymatic asymmetric amination addition is seen as a promising approach for synthesizing amine derivatives, especially unnatural amino acids, which are valuable precursors to fine chemicals and drugs. Despite the broad substrate spectrum of methylaspartate lyase (MAL), some bulky substrates, such as caffeic acid, cannot be effectively accepted. Herein, we report a group of variants structurally derived from Escherichia coli O157:H7 MAL (EcMAL). A combined mutagenesis strategy was used to simultaneously redesign the key residues of the entrance tunnel and binding pocket to explore the possibility of accepting bulky substrates with potential application to chiral drug synthesis. Libraries of residues capable of lining the active center of EcMAL were then constructed and screened by an effective activity solid-phase color screening method using tyrosinase as a cascade catalyst system. Activity assays and molecular dynamics studies of the resultant variants showed that the substrate specificity of EcMAL was modified by adjusting the polarity of the binding pocket and the degree of flexibility of the entrance tunnel. Compared to M3, the optimal variant M8 was obtained with a 15-fold increase in catalytic activity. This structure-based protein engineering of EcMAL can be used to open new application directions or to develop practical multi-enzymatic processes for the production of various useful compounds.

Mechanism of Tethered Agonist-Mediated Signaling by Polycystin-1

Polycystin-1 (PC1) is an important atypical G-protein-coupled receptor (GPCR) with 11 transmembrane domains and its mutations account for 85% of cases of human autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple features with Adhesion GPCRs, including a tethered agonist (TA) from the stalk region that is suggested to activate signaling of the PC1 membrane-embedded C-terminal fragment (CTF). However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined mutagenesis and cellular functional experiments with Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated activation of the wild type, stalkless and ADPKD stalk mutants of the PC1 CTF. Our GaMD simulation findings were highly consistent with the signaling experimental data. Together they revealed an allosteric signaling pathway connecting the stalk, Tetragonal Opening for Polycystins (TOP) and pore loop in activation of the PC1 CTF. Key residue interactions predicted from the GaMD simulations were validated with newly designed mutation-signaling experiments. Our complementary experiments and simulations have provided critical mechanistic insights into the TA-mediated activation of PC1 CTF, which is expected to facilitate rational drug design for the PC1 protein.

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels

ABSTRACTHyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics simulations to show that residue K464 of the C-linker is essential for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that both cAMP and K464E induce a rotation of the intracellular domain relative to the channel pore, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. The adopted poses are in excellent agreement with structural results.