Evolution of a Signaling Nexus Constrained by Protein Interfaces and Conformational States

Heterotrimeric G proteins act as the physical nexus between numerous receptors that respond to extracellular signals and proteins that drive the cytoplasmic response. The Gα subunit of the G protein, in particular, is highly constrained due to its many interactions with proteins that control or react to its conformational state. Various organisms contain differing sets of Gα-interacting proteins, clearly indicating that shifts in sequence and associated Gα functionality were acquired over time. These numerous interactions constrained much of Gα evolution; yet Gα has diversified, through poorly understood processes, into several functionally specialized classes, each with a unique set of interacting proteins. Applying a synthetic sequence-based approach to mammalian Gα subunits, we established a set of seventy-five evolutionarily important class-distinctive residues, sites where a single Gα class is differentiated from the three other classes. We tested the hypothesis that shifts at these sites are important for class-specific functionality. Importantly, we mapped known and well-studied class-specific functionalities from all four mammalian classes to sixteen of our class-distinctive sites, validating the hypothesis. Our results show how unique functionality can evolve through the recruitment of residues that were ancestrally functional. We also studied acquisition of functionalities by following these evolutionarily important sites in non-mammalian organisms. Our results suggest that many class-distinctive sites were established early on in eukaryotic diversification and were critical for the establishment of new Gα classes, whereas others arose in punctuated bursts throughout metazoan evolution. These Gα class-distinctive residues are rational targets for future structural and functional studies

High-Throughput Cryo-EM Enabled by User-Free Preprocessing Routines

Li et al. develop an automatic preprocessing workflow for single-particle cryo-EM. This workflow produces high-resolution particle stacks from micrograph inputs without subjective decision making. They used this workflow to automatically preprocess and analyze six challenging datasets, demonstrating that the workflow correctly identified the high-resolution dataset amid five bad datasets.http://deepblue.lib.umich.edu/bitstream/2027.42/176785/2/High-Throughput Cryo-EM Enabled by User-Free Preprocessing Routines.pdfDescription of High-Throughput Cryo-EM Enabled by User-Free Preprocessing Routines.pdf : Published versio

Structure and function of lysosomal phospholipase A2 and lecithin: cholesterol acyltransferase

Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome

Unveiling the Membrane-Binding Properties of N-Terminal and C-Terminal Regions of G Protein-Coupled Receptor Kinase 5 by Combined Optical Spectroscopies

G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2-31 (GRK5(2-31)) was in random coil whereas GRK5(546-565) was partially helical. When the subphase for the GRK5(2-31) peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK5(2-31) became partially helical. By inspecting the membrane behavior of two different segments of GRK5(2-31), namely, GRK5(2-24) and GRK5(25-31), we found that residues 25-31 are responsible for membrane binding, whereas the helical character is imparted by residues 2-24. With SFG, we deduced that the orientation angle of the helical segment of GRK5(2-31) is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP2 in the model membrane and concluded that the POPC:PIP2 (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca(2+)·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells

Molecular basis activation of lecithin: cholesterol acyltransferase by a compound that increases HDL cholesterol

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients

Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide

Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (~98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (~76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V