The iron-regulated protein FrpD from Neisseria meningitidis is an outer membrane lipoprotein that interacts with very high affinity (Kd ~ 0.2 nM) with the N-terminal domain of FrpC, a Type I-secreted protein from the Repeat in ToXin (RTX) protein family. In the presence of Ca2+, FrpC undergoes Ca2+ -dependent protein trans-splicing that includes an autocatalytic cleavage of the Asp414-Pro415 peptide bond and formation of an Asp414-Lys isopeptide bond. Here, we report the high-resolution structure of FrpD and describe the structure-function relationships underlying the interaction between FrpD and FrpC1-414. We identified FrpD residues involved in FrpC1-414 binding, which enabled localization of FrpD within the low-resolution SAXS model of the FrpD-FrpC1-414 complex. Moreover, the trans-splicing activity of FrpC resulted in covalent linkage of the FrpC1-414 fragment to plasma membrane proteins of epithelial cells in vitro, suggesting that formation of the FrpD-FrpC1-414 complex may be involved in the interaction of meningococci with the host cell surface.

• MeSH
• Bacterial Proteins chemistry   genetics   MeSH
• Cell Adhesion genetics   MeSH
• X-Ray Diffraction MeSH
• Humans MeSH
• Lipoproteins chemistry   metabolism   MeSH
• Membrane Proteins chemistry   genetics   MeSH
• Neisseria meningitidis chemistry   genetics   MeSH
• Periplasmic Binding Proteins chemistry   metabolism   MeSH
• Iron-Binding Proteins chemistry   metabolism   MeSH
• Bacterial Outer Membrane Proteins metabolism   MeSH
• Amino Acid Sequence genetics   MeSH
• Iron chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• frpC protein, Neisseria meningitidis MeSH Browser
• Lipoproteins MeSH
• Membrane Proteins MeSH
• Periplasmic Binding Proteins MeSH
• Iron-Binding Proteins MeSH
• Bacterial Outer Membrane Proteins MeSH
• Iron MeSH

Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity.

• MeSH
• Interferon-gamma chemistry   metabolism   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Models, Molecular * MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon chemistry   genetics   metabolism   MeSH
• Protein Folding * MeSH
• Amino Acid Substitution * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IFNG protein, human MeSH Browser
• Interferon-gamma MeSH
• Receptors, Interferon MeSH

We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor.

• MeSH
• Interferon-gamma chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Surface Plasmon Resonance MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon chemistry   genetics   metabolism   MeSH
• Protein Folding * MeSH
• Molecular Dynamics Simulation * MeSH
• Amino Acid Substitution MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IFNG protein, human MeSH Browser
• Interferon-gamma MeSH
• Receptors, Interferon MeSH