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Intrinsically unstructured domain 3 of hepatitis C Virus NS5A forms a "fuzzy complex" with VAPB-MSP domain which carries ALS-causing mutations - PubMed

Intrinsically unstructured domain 3 of hepatitis C Virus NS5A forms a "fuzzy complex" with VAPB-MSP domain which carries ALS-causing mutations

Garvita Gupta et al. PLoS One. 2012.

Abstract

Hepatitis C virus (HCV) affects nearly 200 million people worldwide and is a leading factor for serious chronic liver diseases. For replicating HCV genome, the membrane-associated replication machinery needs to be formed by both HCV non-structural proteins including NS5A and human host factors. Recently NS5A has been identified to bind ER-anchored human VAP proteins and consequently this interaction may serve as a novel target for design of anti-HCV drugs. So far no biophysical characterization of this interaction has been reported. Here, we dissected the 243-residue VAPB into 4 and 447-residue NS5A into 10 fragments, followed by CD and NMR characterization of their structural properties. Subsequently, binding interactions between these fragments have been extensively assessed by NMR HSQC titration which is very powerful in detecting even very weak binding. The studies lead to three important findings: 1). a "fuzzy complex" is formed between the intrinsically-unstructured third domain (D3) of NS5A and the well-structured MSP domain of VAPB, with an average dissociation constant (Kd) of ~5 µM. 2). The binding-important residues on both NS5A-D3 and VAPB-MSP have been successfully mapped out, which provided experimental constraints for constructing the complex structure. In the complex, unstructured D3 binds to three surface pockets on one side of the MSP structure. Interestingly, two ALS-causing mutations T46I and P56S are also located on the D3-MSP interface. Moreover, NS5A-D3, FFAT-containing proteins and EphA4 appear to have overlapped binding interfaces on the MSP domain. 3). NS5A-D3 has been experimentally confirmed to competes with EphA4 in binding to the MSP domain, and T46I mutation of MSP dramatically abolishes its binding ability to D3. Our study not only provides essential foundation for further deciphering structure and function of the HCV replication machinery, but may also shed light on rationalizing a recent observation that a chronic HCV patient surprisingly developed ALS-like syndrome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CD and NMR characterization of VAPB domains.

(a). Domain organization of the 243-residue human VAPB protein consisting of the major sperm protein (MSP), coiled coil (CC) and transmembrane (TM) domains. (b). Far UV CD spectra of VAPB(1–195) (blue); VAPB(1–150) (green); VAPB(1–125) (brown) and VAPB-CC(151–195) (red). (c). Superimposition of 1H-15N NMR HSQC spectra of VAPB(1–195) (black); VAPB(1–150) (red) and VAPB-CC(151–195) (green). (d). Superimposition of 1H-15N NMR HSQC spectra of VAPB(1–150) (red); VAPB(1–125) (blue).

Figure 2
Figure 2. Identification of VAPB domain binding with NS5A.

(a). Domain organization of the 447-residue HCV NS5A protein consisting of three domains. (b). Prediction of disorder tendency of the full-length NS5A with IUPred server (

http://iupred.enzim.hu/

). (c). Far UV CD spectra of NS5A(33–447) (blue); NS5A-D1(33–202) (green); long NS5A-D2(251–380) (red) and long NS5A-D3(300–447) (brown). (d). Superimposition of 1H-15N NMR HSQC spectra of VAPB(1–195) in the absence of (black) and in the presence of unlabeled NS5A(33–447) (red) at a molar ratio of 1∶2.5 (VAPB/NS5A). (e). Superimposition of 1H-15N NMR HSQC spectra of VAPB-CC(151–195) in the absence of (black) and in the presence of unlabeled NS5A(33–447) (red) at a molar ratio of 1∶2.5 (VAPB-CC/NS5A).

Figure 3
Figure 3. Identification of NS5A domain binding with VAPB.

(a). Superimposition of 1H-15N NMR HSQC spectra of VAPB(1–125) in the absence of (black) and in the presence of unlabeled long NS5A-D3(300–447) at molar ratios of 1∶1.5 (green) and 1∶2.5 (red) (VAPB/NS5A). (b). Superimposition of 1H-15N NMR HSQC spectra of VAPB-CC(151–195) in the absence of (black) and in the presence of unlabeled long NS5A-D3(300–447) at a molar ratio of 1∶4 (red) (VAPB/NS5A). (c). Superimposition of 1H-15N NMR HSQC spectra of VAPB(1–125) in the absence of (black) and in the presence of unlabeled NS5A-D3(359–447) at molar ratio of 1∶1 (green) and 1∶2 (red) (VAPB/NS5A).

Figure 4
Figure 4. Conformational and binding properties of NS5A-D3.

(a). Far UV CD spectrum of NS5A-D3(359–447). (b). Superimposition of 1H-15N NMR HSQC spectra of NS5A-D3(359–447) in the absence of (blue) and in the presence of unlabeled VAPB-MSP(1–125) at molar ratios of 1∶1 (green) and 1∶2 (red) (D3/MSP). (c). Residue-specific 13Cα conformational shift of NS5A-D3(359–447) derived from analysis of triple-resonance heteronuclear NMR spectra including HNCACB and CBCA(CO)NH. Red bars are used to indicate residues undergoing significant shift or disappearance of their HSQC peaks in the presence of unlabeled VAPB-MSP(1–125) at a molar ratio of 1∶2 (D3/MSP).

Figure 5
Figure 5. Conformational and binding properties of NS5A-D3 fragments.

(a). Amino acid sequence of NS5A-D3A, D3B and D3C. (b). Far-UV CD spectra of NS5A-D3 (blue), D3A (red), D3B (brown) and D3C (green). (c). Superimposition of 1H-15N NMR HSQC spectra of NS5A-D3A(394–447) in the absence of (black) and in the presence of unlabeled VAPB-MSP(1–125) at molar ratios of 1∶1 (green) and 1∶2 (red) (D3A/MSP). (d). Residue-specific Hα conformational shift of NS5A-D3A(394–447) derived from analysis of three-dimensional 15N-edited HSQC-TOCSY spectrum. Red bars are used to indicate residues undergoing significant shift or disappearance of their HSQC peaks in the presence of unlabeled VAPB-MSP(1–125) at a molar ratio of 1∶2 (D3A/MSP). (e). Characteristic NOE connectivities of NS5A-D3A(394–447) defining secondary structures derived from analysis of three-dimensional 15N-edited HSQC-NOESY spectrum.

Figure 6
Figure 6. NMR identification of VAPB-MSP residues binding to NS5A-D3 fragments.

Residue-specific changes of integrated 1H and 15N chemical shifts of VAPB-MSP(1–125) in the presence of unlabeled NS5A-D3 (a), D3A (b), D3B (c) and D3C (d) at a molar ratio of 1∶4 (MSP/D3 fragments). Significantly-shifted residues with CSD (chemical shift difference) >1 standard deviations are colored in red and labeled while two regions with disappeared HSQC peaks are indicated by red arrows. (e). Crystal structure of the human VAPB-MSP(1–125) we previously determined (ref. 46) with disappeared (red) and significantly-shifted (yellow) residues mapped out. Two ALS-causing mutants T46I and P56S are displayed in spheres. (f). the FFAT-motif containing ORP1 peptide is further displayed in the structure (ref. 44).

Figure 7
Figure 7. Fitting of chemical shift tracings to obtain dissociation constants (Kd).

Experimental (dots) and fitted (lines) values are shown for the integrated 1H and 15N chemical shift changes of three representative residues: Gln6 (a), Val90 (b) and Asp116 (c) of VAPB-MSP(1–125) induced by gradual addition of NS5A-D3 (red), D3A (green), D3B (purple) and D3C (blue).

Figure 8
Figure 8. Docking model of MSP-D3C complex.

(a). Superimposition of three lowest energy docking models of the MSP-D3C complex. MSP structures are colored in blue and D3C structures in pink. (b). The lowest energy docking model of the MSP-D3C complex, with disappeared and significantly-shifted MSP residues colored in red. Three D3 regions critical for binding with MSP are displayed in spheres. (c). The lowest energy docking model of the MSP-D3C complex, with MSP structure displayed in surface and three discrete D3 regions critical for binding with MSP are displayed in spheres of different colors. Three MSP surface pockets are labeled as P1, P2 and P3 respectively. (d). The lowest energy docking model of the MSP-D3C complex, with the MSP electrostatic potential displayed, with blue, red and grey corresponding to positive, negative and neutral potential values. (e). Hydrogen bonds between D3C and MSP in the complex. Only the residues having the interfacial hydrogen bonds are displayed in sticks and the hydrogen bonds are indicated by the red dashed lines.

Figure 9
Figure 9. NS5A competes with EphA4 in binding with MSP.

(a). Superimposition of 1H-15N NMR HSQC spectra of VAPB-MSP(1–125) saturated with the pre-existence of D3C at a molar ratio of 1∶2 (MSP/D3C), in the absence of (blue) and in the presence of 181-residue EphA4 ligand binding domain at a molar ratio of 1∶10 (red) (MSP/EphA4). Significantly-shifted residues are labeled. (b). Crystal structure of the human VAPB-MSP(1–125) with significantly-shifted residues displayed in red spheres. (c). Residue-specific changes of integrated 1H and 15N chemical shifts of VAPB-MSP(1–125) T46I mutant in the presence of unlabeled NS5A-D3C at a molar ratio of 1∶6 (MSP/D3C). Significantly-shifted residues with CSD (chemical shift difference) >1 standard deviation are colored in red.

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