A recurring packing contact in crystals of InlB pinpoints functional binding sites in the internalin domain and the B repeat

Christina Geerds,Timo Meyer,Christiane Widmann,Willem M Bleymüller,Hartmut H Niemann

doi:10.1107/s2059798322000432

Christina Geerds, Timo Meyer + Show 3 more

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https://doi.org/10.1107/s2059798322000432

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Abstract

InlB, a bacterial agonist of the human receptor tyrosine kinase MET, consists of an N-terminal internalin domain, a central B repeat and three C-terminal GW domains. In all previous structures of full-length InlB or an InlB construct lacking the GW domains (InlB392), there was no interpretable electron density for the B repeat. Here, three InlB392 crystal structures in which the B repeat is resolved are described. These are the first structures to reveal the relative orientation of the internalin domain and the B repeat. A wild-type structure and two structures of the T332E variant together contain five crystallographically independent molecules. Surprisingly, the threonine-to-glutamate substitution in the B repeat substantially improved the crystallization propensity and crystal quality of the T332E variant. The internalin domain and B repeat are quite rigid internally, but are flexibly linked to each other. The new structures show that inter-domain flexibility is the most likely cause of the missing electron density for the B repeat in previous InlB structures. A potential binding groove between B-repeat strand β2 and an adjacent loop forms an important crystal contact in all five crystallographically independent chains. This region may represent a hydrophobic `sticky patch' that supports protein-protein interactions. This assumption agrees with the previous finding that all known inactivating point mutations in the B repeat lie within strand β2. The groove formed by strand β2 and the adjacent loop may thus represent a functionally important protein-protein interaction site in the B repeat.

Highlights

InlB is an invasion protein that is located on the surface of the bacterium Listeria monocytogenes (Dramsi et al, 1995; Lingnau et al, 1995)
The T332E substitution is one of two point mutations that we had previously found to have a negative effect on the biological function of the B repeat (Bleymuller et al, 2016)
The circular-dichroism spectrum and the elution behaviour on a gel-filtration column confirmed this assumption, as they were basically identical for the wild-type B repeat and the T332E variant (Bleymuller et al, 2016)

Summary

Introduction

InlB is an invasion protein that is located on the surface of the bacterium Listeria monocytogenes (Dramsi et al, 1995; Lingnau et al, 1995). Binding of InlB to the receptor tyrosine kinase MET on host cells is one way to induce internalization of bacteria (Shen et al, 2000). MET acts as a receptor for hepatocyte growth factor/scatter factor (HGF/SF) and is essential during mammalian development (Birchmeier et al, 2003). Cellular phenotypes in response to InlB stimulation resemble those observed upon MET activation by HGF/SF. They include cell motility (for example cell scatter of clonally growing MDCK or HT-29 cells) and cell proliferation (Shen et al, 2000; Niemann et al, 2007).

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