Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose

During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the high-flying bar-headed goose (Anser indicus), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. To test the effects of observed substitutions on evolutionarily relevant genetic backgrounds, we synthesized all possible genotypic intermediates in the line of descent connecting the wildtype barheaded goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed one major-effect mutation that significantly increased Hb-O2 affinity on all possible genetic backgrounds. Two other mutations exhibited smaller average effect sizes and less additivity across backgrounds. One of the latter mutations produced a concomitant increase in the autoxidation rate, a deleterious side-effect that was fully compensated by a second-site mutation at a spatially proximal residue. The experiments revealed three key insights: (i) subtle, localized structural changes can produce large functional effects; (ii) relative effect sizes of functionaltering mutations may depend on the sequential order in which they occur; and (iii) compensation of deleterious pleiotropic effects may play an important role in the adaptive evolution of protein function

Unraveling the photoactivation mechanism of a light activated adenylyl cyclase using ultrafast spectroscopy coupled with unnatural amino acid mutagenesis

The hydrogen bonding network that surrounds the flavin in Blue Light Utilizing FAD (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. The network contains a highly conserved tyrosine that is essential for photoactivation. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid (UAA) incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase (AC) from Oscillatoria Acuminata (OaPAC). Our work elucidates the direct connection between the photoactivation process in the BLUF domain and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate formation of FADH● as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogs to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FAD● ̶. The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the AC domain. While the 3F-Y6 and 2,3-F2Y6 variants undergo the complete photocycle and catalyze the conversion of ATP to cAMP, enzyme activity is abolished in the 3,5-F2Y6 and 2,3,5-F3Y6 variants where the photocycle is halted at FAD● ̶. Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in adenylyl cyclase activity

Structural insights into the HBV receptor and bile acid transporter NTCP

B型肝炎ウイルスの受容体“胆汁酸輸送体”の立体構造を解明. 京都大学プレスリリース. 2022-05-18.Roughly 250 million people are infected with hepatitis B virus (HBV) worldwide, and perhaps 15 million also carry the satellite virus HDV, which confers even greater risk of severe liver disease. Almost ten years ago the HBV receptor was identified as NTCP (sodium taurocholate co-transporting polypeptide), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large (L) protein. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria, and these models with ten transmembrane helices are believed to resemble strongly both NTCP and ASBT. Using cryo-electron microscopy we have solved the structure of NTCP bound to an antibody, clearly showing the transporter has no equivalent to the first transmembrane helix of other SLC10 models, leaving the N-terminus exposed on the extracellular face. Comparison of the different structures indicates a common mechanism of bile acid transport, but the NTCP structure also displays a pocket formed by residues known to interact with preS1, presenting new and enticing opportunities for structure-based drug design

The crystal structure and oligomeric form of Escherichia coli L,D-carboxypeptidase A

Bacterial peptidoglycan is constructed by cross-linking sugar chains carrying pentapeptide building blocks with two d-alanine residues at the C-terminus. Incorporation into the polymer and subsequent breakdown of peptidoglycan releases a tetrapeptide with a single d-alanine residue. Removal of this residue is necessary for the tripeptide to receive a new D-Ala-D-Ala dipeptide in the synthetic pathway, but proteases are generally unable to work with substrates having residues of unusual chirality close to the scissile bond. Processing of the tetrapeptide is carried out by a dedicated ld-carboxypeptidase, which is of interest as a novel drug target. We describe the high resolution crystal structure of the enzyme from E. coli, and demonstrate the dimeric structure is highly conserved

Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose

During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the high-flying bar-headed goose (Anser indicus), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. To test the effects of observed substitutions on evolutionarily relevant genetic backgrounds, we synthesized all possible genotypic intermediates in the line of descent connecting the wildtype barheaded goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed one major-effect mutation that significantly increased Hb-O2 affinity on all possible genetic backgrounds. Two other mutations exhibited smaller average effect sizes and less additivity across backgrounds. One of the latter mutations produced a concomitant increase in the autoxidation rate, a deleterious side-effect that was fully compensated by a second-site mutation at a spatially proximal residue. The experiments revealed three key insights: (i) subtle, localized structural changes can produce large functional effects; (ii) relative effect sizes of functionaltering mutations may depend on the sequential order in which they occur; and (iii) compensation of deleterious pleiotropic effects may play an important role in the adaptive evolution of protein function

Structural Basis of Sequential and Concerted Cooperativity

Allostery is a property of biological macromolecules featuring cooperative ligand binding and regulation of ligand affinity by effectors. The definition was introduced by Monod and Jacob in 1963, and formally developed as the “concerted model” by Monod, Wyman, and Changeux in 1965. Since its inception, this model of cooperativity was seen as distinct from and not reducible to the “sequential model” originally formulated by Pauling in 1935, which was developed further by Koshland, Nemethy, and Filmer in 1966. However, it is difficult to decide which model is more appropriate from equilibrium or kinetics measurements alone. In this paper, we examine several cooperative proteins whose functional behavior, whether sequential or concerted, is established, and offer a combined approach based on functional and structural analysis. We find that isologous, mostly helical interfaces are common in cooperative proteins regardless of their mechanism. On the other hand, the relative contribution of tertiary and quaternary structural changes, as well as the asymmetry in the liganded state, may help distinguish between the two mechanisms

Crystal structure of penicillin binding protein 4 (dacB) from Escherichia coli, both in the native form and covalently linked to various antibiotics

The crystal structure of penicillin binding protein 4 (PBP4) from Escherichia coli, which has both DD-endopeptidase and DD-carboxypeptidase activity, is presented. PBP4 is one of 12 penicillin binding proteins in E. coli involved in the synthesis and maintenance of the cell wall. The model contains a penicillin binding domain similar to known structures, but includes a large insertion which folds into domains with unique folds. The structures of the protein covalently attached to five different antibiotics presented here show the active site residues are unmoved compared to the apoprotein, but nearby surface loops and helices are displaced in some cases. The altered geometry of conserved active site residues compared with those of other PBPs suggests a possible cause for the slow deacylation rate of PBP4