Invited review GPCR structural characterization: Using fragments as building blocks to determine a complete structure

Abstract

The structural characterization of G protein-coupled receptors has surged since the development of methodologies to facilitate the crystallization of these highly helical, seven transmembrane, integral membrane receptors. In the past seven years, eighteen GPCR structures were determined by X-ray crystallography. The crystal structures represent a static picture of these conformationally flexible signal transducers. Analyses that probe their dynamics and conformational changes require other techniques, in particular solution state nuclear magnetic resonance studies. Such investigations are challenged by the size of GPCRs, their α-helical structure, which limits resonance dispersion, their tendencies to aggregate in micellar preparations and their conformational heterogeneity. For many years, groups have been studying GPCR fragments as a means to overcome some of these difficulties. The results of these fragment analyses are presented here. Review of the literature reveals that much of the original work depended on circular dichroism, infra-red spectroscopy and fluorescence approaches. High resolution structures obtained by NMR are compared, where applicable, to the available crystal structures. In most cases, the work done on fragments by biophysical analysis is validated by these comparisons. Our perspective on the field of GPCR fragment analysis is presented together with the future goals that must be considered if work with fragments is continued.

A Paradigm for Peptide Hormone-GPCR Analyses

“…These studies required the development of synthetic and biosynthetic approaches to prepare various Ste2p domains. Much of this work has been previously summarized [ 81 , 82 , 83 ] and will not be discussed in detail. In the following section, we will discuss primarily what we learned by studies of large fragments of a GPCR, and how this work may have contributed to understanding the structure of the intact receptor.…”

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

“…This fragment is larger (by at least one TM segment) than the vast majority of other GPCR fragments that have been produced and studied to date (Cohen et al 2014). Biophysical studies in an organic solvent/water mixture (50% HFIP in water) and in SDS micelles indicate that this GPCR fragment has appropriate secondary structuring and is suitable for long-term characterization by solutionstate NMR spectroscopy.…”

“…This species migrates consistently with the AR_TM1-3 protein in cell pellet and mass spectrometry has consistently shown only the monomeric species (Figure 5). Based on previous studies of SDS micelle-solubilized TM domain oligomerization (Tulumello and Deber 2009) and the observation of oligomers of a larger yeast GPCR fragment by SDS-PAGE (Cohen et al 2014;Potetinova et al 2012), it is likely that any oligomeric state in SDS micelles would be retained during SDS-PAGE. Finally, the observed line widths and suitability of standard HSQC experiments rather than relaxation optimized experiments such as TROSY (Cavanagh et al 2007) are consistent with AR_TM1-3 being monomeric in both 50% HFIP and SDS micelles.…”

“…This model justifies the study of fragments comprised of single or multiple TM segments, the so-called "divide and conquer" approach, as it states that an isolated TM helix represents a distinct folding subunit that can be separated from its parent protein without losing its inherent structural characteristics (Bordag and Keller 2010). Beyond general applicability to membrane proteins when judiciously applied (Bordag and Keller 2010), this approach has been quite extensively applied for studies of GPCRs (Cohen et al 2014). …”

“…As a result of the difficulties in expression of isotope-labeled full-length GPCRs, fragments of receptors ranging in size from 20-120 amino acids have been studied (Langelaan et al 2013;Tiburu et al 2011;Tikhonova and Costanzi 2009;Tyukhtenko et al 2009;Zhao et al 2006;Zou et al 2008). Ste2p, a yeast GPCR, is the only example where larger fragments (4 and 5 transmembrane (TM) segments) have been reported for structural characterization by NMR spectroscopy (Cohen et al 2014;Potetinova et al 2012). …”