Design and analysis of post-fusion 6-helix bundle of heptad repeat regions from Newcastle disease virus F protein

Abstract

Fusion of paramyxovirus to the cell involves receptor binding of the HN glycoprotein and a number of conformational changes of F glycoprotein. The F protein is expressed as a homotrimer on the virus surface. In the present model, there are at least three conformations of F protein, i.e. native form, pre-hairpin intermediate and the post-fusion state. In the post-fusion state, the two highly conserved heptad repeat (HR) regions of F protein form a stable 6-helix coiled-coil bundle. However, no crystal structure is known for this state for the Newcastle disease virus, although the crystal structure of the F protein native form has been solved recently. Here we deployed an Escherichia coli in vitro expression system to engineer this 6-helix bundle by fusion of either the two HR regions (HR1, linker and HR2) or linking the 6-helix [3 x (HR1, linker and HR2)] together as a single chain. Subsequently, both of them form a stable 6-helix bundle in vitro judging by gel filtration and chemical cross-linking and the proteins show salient features of an alpha-helix structure. Crystals diffracting X-rays have been obtained from both protein preparations and the structure determination is under way. This method could be used for crystallization of the post-fusion state HR structures of other viruses.

Interacting domains of the HN and F Proteins of paramyxovirus

“…Some studies showed that the HN protein associates with the F protein via specific interaction between the heptad repeat regions of both proteins. For Avian parainfluenza virus-1 (APIV-1, NDV), the head region (HNH) might interact with the HR2 region [8] , and HR2 might interact with HR1 [9] . For Avian parainfluenza virus-1 (APIV-1, NDV), the head region (HNH) might interact with the HR2 region [8] , and HR2 might interact with HR1 [9] .…”

The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor

“…In addition, the structure of a substrate-analogue inhibitor complex shows an unexpected mode of inhibitor binding and provides structure basis for rational drug design. , was purified by GST-glutathione affinity chromatography, cleaved with GST-rhinovirus 3C protease (11), and the recombinant SARS-CoV M pro was further purified by using anion-exchange chromatography. The purified and concentrated SARS-CoV M pro (10 mg͞ml) was stored in 10 mM Tris⅐HCl, pH 7.5͞1 mM DTT͞1 mM EDTA.…”

Interacting domains of the HN and F Proteins of paramyxovirus

“…The conserved stalk domain (the HR region) is critical to the role of HN in the promotion of fusion and may be directly involved in its interaction with the F protein HR regions [7] . For Avian parainfluenza virus-1 (APIV-1, NDV), the head region (HNH) might interact with the HR2 region [8] , and HR2 might interact with HR1 [9] . Therefore the relationships among the HNH, HR1 and HR2 are the key questions in the research of the HN-F interaction domains.…”

“…We chose a BLAST (http://www.ncbi.nlm.nih.gov/ BLAST) analysis program in identifying HNH of APIV-2 HN protein and HR1 and HR2 regions of F protein by comparing them with relative regions of APIV-1 published recently [8,9] . Meanwhile, we chose the LearnCoil-VMF (http://nightingale.lcs.mit.edu) program to identify F protein heptad repeat coiled coils.…”