2.1. Interfaces revealed by UAA incorporation and crosslinking
Incorporation of a photo-reactive UAA into a target protein in live
cells followed by controlled crosslinking in situ provides a
great advantage of identifying target protein-specific binding
partner(s) in a native environment (Figure 2A). Importantly, it
circumvents the need for target protein extraction and purification in
aqueous solution before crosslinking, which is almost impossible for
some proteins, especially those in cellular membranes like G
protein-coupled receptors (GPCRs). GPCRs are the largest family of
integral membrane proteins with a characteristic structure comprising
seven transmembrane helix folds, three intracellular loops together with
the N-terminus, and three extracellular loops together with the
C-terminus (Cvicek et al., 2016). Membranous and flexible natures of
GPCRs usually compromise the sample preparation for classical structure
analyses that require a substantial sample amount and purity. Even
though dozens of GPCR structures have been determined, most of them are
believed to represent a single snapshot of GPCR that otherwise would
wiggle vigorously in a native environment.
CXC chemokine receptor 4 (CXCR4) is a pathologically and clinically
important GPCR in cancers, inflammation, and viral infection. To probe
the binding site of T140, a 14-residue cyclic peptide HIV-1 entry
blocker, CXCR4 was site-specifically mutated with a photoactivatable
UAA, p-benzoyl-l-phenylalanine (BzF), at various positions one-by-one in
live cells (Grunbeck et al., 2011). Co-incubation of the fluorescently
labeled T140 with CXCR4-transfected cells was followed by UV exposure to
trap a CXCR4-T140 complex. A series of experiments for each CXCR4 mutant
showed that the crosslinked complex was formed only when BzF had been
incorporated at Phe189. Based on a characteristic 3 Å-long reactivity
distance of BzF as well as the positional information gained from the
crosslinking analyses, molecular modeling could successfully derive a
more accurate model of the CXCR4-T140 interface modified from available
crystal structures. Similarly, strategic incorporation of
photo-crosslinking UAA allowed identification of contact residues of
CCR5 in complex with a small molecule HIV-1 entry inhibitor, maraviroc
(Grunbeck et al., 2012). Newly discovered contact residues previously
not recognized in the computational modeling could serve as a cue to
reconstruct the interface taking the dynamic allosteric binding of
maraviroc into account (Grunbeck et al., 2012). Although the
UAA-mediated crosslinking studies mentioned above lacked the mass
spectrometric analysis, a genetically well-defined position of
crosslinking and the ability to incorporate the UAA in any position of
interest could make a significant contribution to remodeling GPCR-ligand
structures to display the biological interface. The most attractive
aspect of the method in studying the structural biology of GPCRs lies in
that important contact sites can be identified even when GPCR behaves
dynamically in a native cellular environment in response to different
biological stimulation. By analyzing 25 different mutants of the
angiotensin II type 1 receptor (AT1R) containing a photo-reactive UAA,
Gagnon et al. demonstrated that AT1R exhibits structurally differential
binding modes, i.e., distinct conformations or residues, with an
intracellular β-arrestin regulator depending on the presence of an
extracellular ligand, angiotensin II (Gagnon et al., 2019).
Photo-reactive UAA incorporation technique in live cells readily allows
investigation of a dynamic interface hidden in the transmembrane domain
which is much more challenging to recombinantly prepare in vitrothan the extracellular domain. CGRP is a ligand that binds to the
CLR/RAMP1 receptor. While the crystal structure of the extracellular
domain of CLR/RAMP1 in complex with CGRP and corresponding contact
residues had been available, the interface located in the transmembrane
domain remained to be probed. Crosslinked residue screening by
photo-reactive UAA crosslinker incorporation at multiple potential
contact sites in the CLR transmembrane helix domain identified major
contact residues, providing insight into the extent of CGRP penetration
into the transmembrane core of CLR (Simms et al., 2018). It should be
noted that, besides GPCR, most protein complexes recombinantly
expressible in E. coli or mammalian cells can benefit from techniques
reviewed above for investigation of biological interfaces regardless of
their size, cellular location, and complexity (Bridge et al., 2019;
Owens et al., 2019; Rubino et al., 2020).
Since biochemical data obtained from the UAA-mediated crosslinking are
an array of band shift patterns resulting from various
crosslinked-target proteins or fragments in the immunoblotted gel
electrophoresis, the output is generally moderate or low in
three-dimensional conformation and resolution. The crosslinking data can
be best utilized when integrated with other structural information to
give a refined structure with high resolution and fidelity. For example,
the class B corticotropin-releasing factor receptor type 1 (CRF1R) was
mapped by genetically incorporating an UAA crosslinker systematically to
characterize the difference in a conformational change and an
interfacial landscape, when CRF1R was stimulated by an agonist or
antagonist. Differences in band shift patterns were evident in western
blotting, and the inter-residue distance constraints, a characteristic
of the crosslinked chemical structure, estimated for all of the
crosslinked residues were applied to computationally generate
conformational models of the agonist- and the antagonist-bound CRF1R
complex. Extensive sampling of conformations led to optimized structure
models for both complexes, revealing that CRF1R adopted distinct binding
interfaces and local conformations to engage the agonist and the
antagonist, respectively (Seidel et al., 2017). Remarkably, the
predicted models have been found to be very compatible with the
high-resolution cryo-EM structures obtained years later (Liang et al.,
2020; Ma et al., 2020). A unique hormone-binding motif of the insulin
receptor could be recognized from the crosslinking data in conjunction
with the preexisting crystal structure of the apo-insulin receptor. As
was expected from the crystal contacts, a photo-reactive UAA
incorporated in the typical β-strands 2 and 3 in the L1 domain of the
insulin receptor α-subunit was found to be crosslinked to the other
α-subunit in an apo-state. However, in a holo-state where the insulin
receptor was bound to the insulin, the crosslinks were made to the
insulin, implying a dynamic interface change caused by the induced fit
of the insulin (Whittaker et al., 2012). Similarly, a recent study
performed a crystal structure-based photo-crosslinking analysis of the
macromolecular complex of nuclear pore proteins, Nup82, Nup116, and
Nup159. Of note, crosslinking patterns demonstrated that, in comparison
to the crystal interfaces, the interfaces of Nup82 in contact with
Nup116 and 159 were significantly different in areas and residues
involved (Shin and Lim 2020). Interestingly, some contact residues in
the interface engaging Nup82 and Nup116 in the ternary complex seemed to
lose contacts when Nup82 and Nup116 formed the binary complex by
themselves without Nup159, indicating a dynamic nature of the interface
not locally isolated but wriggling in conjunction with overall
structural changes. Abovementioned and related studies are summarized in
the header row of ‘photo-crosslinking UAA in live cells’ (Table 1).