Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli.

Crystallography has advanced our understanding of G protein-coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type-like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein-coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein-coupled receptor signaling, receptor maturation, and desensitization.

Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations.

The structural features determining efficient biosynthesis, stability in the membrane and, after solubilization, in detergents are not well understood for integral membrane proteins such as G protein-coupled receptors (GPCRs). Starting from the rat neurotensin receptor 1, a class A GPCR, we generated a separate library comprising all 64 codons for each amino acid position. By combining a previously developed FACS-based selection system for functional expression [Sarkar C, et al. (2009) Proc Natl Acad Sci USA 105:14808-14813] with ultradeep 454 sequencing, we determined the amino acid preference in every position and identified several positions in the natural sequence that restrict functional expression. A strong accumulation of shifts, i.e., a residue preference different from wild type, is detected for helix 1, suggesting a key role in receptor biosynthesis. Furthermore, under selective pressure we observe a shift of the most conserved residues of the N-terminal helices. This unique data set allows us to compare the in vitro evolution of a GPCR to the natural evolution of the GPCR family and to observe how selective pressure shapes the sequence space covered by functional molecules. Under the applied selective pressure, several positions shift away from the wild-type sequence, and these improve the biophysical properties. We discuss possible structural reasons for conserved and shifted residues.

Directed evolution of G-protein-coupled receptors for high functional expression and detergent stability.

G-protein-coupled receptors (GPCRs) are cell-surface receptors exhibiting a key role in cellular signal transduction processes, thus making them pharmacologically highly relevant target proteins. However, the molecular mechanisms driving receptor activation by ligand binding and signal transduction are poorly understood, since as integral membrane proteins, most GPCRs are very challenging for functional and structural studies. The biophysical properties of natural GPCRs, usually required by the cell in only low amounts, support their functionality in the lipid bilayer but are insufficient for high-level recombinant overexpression and stability in detergent solution. Current structural information about GPCRs is thus limited to a subset of GPCRs with either intrinsically favorable or properly improved biophysical behavior. Recently, directed protein evolution techniques for functional expression and detergent stability have been developed to increase the accessibility of GPCRs for functional and structural studies. Directed evolution does not rely on any preconceived notion of what might be limiting biophysical properties. By random mutagenesis combined with a high-throughput screening and selection system, directed protein evolution has the power to efficiently isolate rare phenotypes and thus contribute to the elucidation of the stability-determining factors, in addition to solving the practical problem of creating stable GPCRs. In the current chapter, protocols for generation of genetic diversity within GPCRs and selection are provided and discussed.

Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution.

To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution.