Random Mutagenesis of the Complement Factor 5a (C5a) Receptor N Terminus Provides a Structural Constraint for C5a Docking

Hagemann, Ian S.; Narzinski, Kirk; Floyd, Desiree H.; Baranski, Thomas J.

doi:10.1074/jbc.m607686200

Cited by 16 publications

(31 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Library Construction and Screening-Libraries of mutant C5aRs were generated as described previously (12,23). Briefly, the nucleotide sequence of the region of interest (NT or EC2) was flanked by silent restriction sites, then replaced by a noncoding "stuffer" sequence to prevent wild-type contamination of the library.…”

Section: Methodsmentioning

confidence: 99%

“…2A). The seventh cysteine (Cys 27 ) has an outwardly directed side chain (29,30) that was previously used for disulfide trapping analysis (12). The data in our earlier study showed that in the receptor-ligand complex, Cys 27 of C5a is positioned close to residues 24 -30 of the C5aR.…”

Section: Generation Of Mutant C5a Ligands With Unpairedmentioning

confidence: 97%

“…In each of these bait mutants, Cys 27 was replaced with Arg; this residue was chosen because it replaces Cys 27 in rodent C5a sequences, which are otherwise highly conserved relative to human. The sites in the C5a ligand were chosen based on their surface accessibility, previous data demonstrating potential roles in receptor binding, and inspection of our initial models of the docked C5a ligand (12). To verify that the ligands were functional, we coexpressed them with the C5aR in a S. cerevisiae expression system with a LacZ (␤-galactosidase) reporter.…”

Section: Generation Of Mutant C5a Ligands With Unpairedmentioning

confidence: 99%

“…Furthermore, NMR spectroscopy has shown that the structure of the isolated C5aR N terminus is altered by incubation with C5a, suggesting that the N terminus of the receptor interacts with the ligand (11). Because hexapeptide analogs such as W5Cha are full agonists for the C5aR, the first site is apparently not a key component of the switch mechanism (8,(11)(12)(13)(14). Instead, the first site confers "address"-like specificity on the interaction.…”

mentioning

confidence: 99%

“…Rather, the affinity is built from multiple individually weak interactions (12). In our random mutagenesis experiments, mutations were introduced into multiple positions within the receptor region of interest; receptors that retained signaling ability despite their multiple mutations were selected by a functional screen in Saccharomyces cerevisiae.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

Hagemann

Miller

Klco

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.One of the most biologically important families of receptors is the G protein-coupled receptors (GPCRs), 2 which eukaryotic cells use to sense signals as diverse as light, odorants, small molecules, and polypeptide hormones (1). The GPCRs, which number ϳ1000 in the human genome (2), have a shared topology made up of seven transmembrane domains (TMs) linked by intracellular and extracellular (EC) loops, along with an extracellular N terminus and intracellular C terminus (Fig. 1). These receptors also have a shared mechanism of action, whereby the activated receptor serves as a guanosine exchange factor on the ␣ subunit of a heterotrimeric G protein, thus transmitting the signal to the interior of the cell. Many drugs are directed against GPCRs, including adrenergic, muscarinic, dopaminergic, serotonergic, GABAergic, and histaminergic receptors. Taken together, drugs acting on GPCRs make up an estimated 50% of the current pharmacopoeia (3).The structural basis of receptor-ligand interaction is of great interest in both basic and applied pharmacology. In the case of GPCRs, a central question is how receptors with a single common topology can be activated by such a wide variety of ligands. A related question is how specificity is built into these receptors, so that each is activated only by the appropriate ligands.Many GPCRs are activated by polypeptide ligands, including chemokines such as SDF-1 (CXCL12), physiologic modulators such as angiotensin II, glycoprotein hormones such as luteinizing hormone, and chemotactic factors such as complement factor 5a (C5a). These ligands are too large to fit entirely in an interhelical cleft of a GPCR, so their bulk must serve some adaptive function other than receptor activation per se. In some cases, the ligand is recruited by its affinity for the receptor extracellular domains, and a second discret...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Generation Of Mutant C5a Ligands With Unpairedmentioning

confidence: 97%

Section: Generation Of Mutant C5a Ligands With Unpairedmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

Hagemann

Miller

Klco

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Interaction of Human C5a with the Major Peptide Fragments of C5aR1: Direct Evidence in Support of “Two-Site” Binding Paradigm

2021

View full text Add to dashboard Cite

The C5a receptor’s (C5aR1) physiological function in various tissues depends on its high-affinity binding to the cationic proinflammatory glycoprotein C5a, produced during the activation of the complement system. However, an overstimulated complement can quickly alter the C5a–C5aR1 function from physiological to pathological, as has been noted in the case of several chronic inflammation-induced diseases like asthma, lung injury, multiorgan failure, sepsis, and now COVID-19. In the absence of the structural data, the current study provides the confirmatory biophysical validation of the hypothesized “two-site” binding interactions of C5a, involving (i) the N-terminus (NT) peptide (“Site1”) and (ii) the extracellular loop 2 (ECL2) peptide of the extracellular surface (ECS) of the C5aR1 (“Site2”), as illustrated earlier in the reported model structural complex of C5a–C5aR1. The biophysical and computational data elaborated in the study provides an improved understanding of the C5a–C5aR1 interaction at an atomistic resolution, highlighting the energetic importance of the aspartic acids on the NT-peptide of C5aR1 toward binding of C5a. The current study can potentially advance the search and optimization of new-generation alternative “antibodies” as well as “neutraligands” targeting the C5a to modulate its interaction with C5aR1.

show abstract

Modeling Molecular Mechanisms of Binding of the Anaphylatoxin C5a to the C5a Receptor

2008

Self Cite

View full text Add to dashboard Cite

This study presents the 3D model of the complex between the anaphylatoxin C5a and its specific receptor, C5aR. This is the first 3D model of a G-protein-coupled receptor (GPCR) complex with a peptide ligand deduced by a molecular modeling procedure analyzing various conformational possibilities of the extracellular loops and the N-terminal segment of the GPCR. The modeling results indicated two very different ways of interacting between C5a and C5aR at the two interaction sites suggested earlier based on the data of site-directed mutagenesis. Specifically, C5a and C5aR can be involved in "mutual-induced fit", where the interface between the molecules is determined by both the receptor and the ligand. The rigid core of the C5a ligand selects the proper conformations of the highly flexible N-terminal segment of C5aR (the first interaction site). At the same time, the binding conformation of the flexible C-terminal fragment of C5a is selected by well-defined interactions with the TM region of the C5aR receptor (the second interaction site). The proposed 3D model of C5a/C5aR complex was built without direct use of structural constraints derived from site-directed mutagenesis reserving those data for validation of the model. The available data of site-directed mutagenesis of C5a and C5aR were successfully rationalized with the help of the model. Also, the modeling results predicted that the full-length C5a and C5a-des74 metabolite would have different binding modes with C5aR. Modeling approaches employed in this study are readily applicable for studies of molecular mechanisms of binding of other polypeptide ligands to their specific GPCRs.

show abstract

Random Mutagenesis of the Complement Factor 5a (C5a) Receptor N Terminus Provides a Structural Constraint for C5a Docking

Cited by 16 publications

References 51 publications

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

Interaction of Human C5a with the Major Peptide Fragments of C5aR1: Direct Evidence in Support of “Two-Site” Binding Paradigm

Modeling Molecular Mechanisms of Binding of the Anaphylatoxin C5a to the C5a Receptor

Contact Info

Product

Resources

About