Pre‐structured motifs in the natively unstructured preS1 surface antigen of hepatitis B virus

Abstract

The preS1 surface antigen of hepatitis B virus (HBV) is known to play an important role in the initial attachment of HBV to hepatocytes. We have characterized structural features of the full-length preS1 using heteronuclear NMR methods and discovered that this 119-residue protein is inherently unstructured without a unique tertiary structure under a nondenaturing condition. Yet, combination of various NMR parameters shows that the preS1 contains ''pre-structured'' domains broadly covering its functional domains. The most prominent domain is formed by residues 27-45 and overlaps with the putative hepatocyte-binding domain (HBD) encompassing residues 21-47, within which two welldefined pre-structured motifs, formed by Pro 32 -Ala 36 and Pro 41 -Phe 45 are found. Additional, somewhat less prominent, pre-structured motifs are also formed by residues 11-18, 22-25, 37-40, and 46-50. Overall results suggest that the preS1 is a natively unstructured protein (NUP) whose N-terminal 50 residues, populated with multiple pre-structured motifs, contribute critically to hepatocyte binding.Keywords: hepatitis B virus; preS1; NMR; natively unstructured protein; pre-structured motif Supplemental material: see www.proteinscience.org Hepatitis B virus (HBV) is a member of the hepadnaviridae family (Ganem and Varmus 1987;Chisari et al. 1989) that poses a significant health threat to millions of people worldwide, particularly in Africa, Asia, and certain parts of Europe (Fung and Lok 2004;Wright 2006). Whereas HBV infection itself may not appear so fatal as AIDS or SARS chronic hepatitis B infection often develops into more serious symptoms such as cirrhosis, liver failure, and hepatocelluar carcinoma (Blumberg et al. 1989). While successful administration of HBV vaccines led to a significant reduction of HBV-related casualties (Stephenne 1990;Mahoney et al. 1993;Jilg 1998;Poland and Jacobson 2004), emergence of escape mutants or nonresponders to current vaccines argues for attempts to develop vaccines with better efficacy. In addition, more specific anti-HBV therapeutics are needed in order to completely eradicate HBV infection, since nucleoside drugs currently in use for HBV-related symptoms are not HBV specific, often causing side effects or inducing drug resistance. Efforts to discover anit-HBVspecific agents based upon natural products or siRNAs have been met with only partial success (Saag 2006;Shin et al. 2006).Even though it is generally accepted that HBV infection begins with an attachment of HBV surface antigens to a HBV-specific hepatocyte receptor, followed by subsequent entry of viral DNA into hepatocyte (Neurath 3 These authors contributed equally to this work. Reprint requests to: Kyou-Hoon Han, Molecular Cancer Research Center, Division of Molecular Therapeutics, KRIBB, Daejeon 305-806, Korea; e-mail: khhan600@kribb.re.kr; fax: 82-42-860-4259.Abbreviations: HBV, hepatitis B virus; NMR, nuclear magnetic resonance; NUP, natively unstructured protein; CSI, chemical-shift index.Article published online ahead of pr...

Broadly neutralizing anti‐HBV antibody binds to non‐epitope regions of preS1

“…Taken together with the NMR data, the results indicate that all the low-affinity binding sites are largely unstructured. As probed by the previous NMR analysis on the full-length preS1, there was no long range NOE between the major epitope and the low-affinity sites [30], indicating that the multiple low-affinity binding sites are not clustered in close proximity to the major epitope.…”

“…The concentrationdependent increase of RU signals clearly confirms that there are specific interactions between the Fab and all the epitope-near preS1 regions. The interactions of these four preS1 peptides for the Fab were weak with the equilibrium dissociation constant (K d ) values of 1-2 mM range: 1.54, 1.63, 2.04, and 1.99 mM for preS1 (1-11), preS1 (27)(28)(29)(30)(31)(32)(33)(34)(35), preS1 (66-76), and preS1 (95-104), respectively. Although this binding affinity seems low, it is comparable to the affinities observed for germline antibody binding [28] and TCR-peptide/MHC (pMHC) interactions [35].…”

“…HzKR127 Fab in 25 mM sodium acetate, pH 5.5 was immobilized to a channel of CM5 sensor chip using an amine coupling kit (BIAcore AB) and the control flow-subtracted sensograms are obtained. The peptide concentrations range from 10 to 640 nM for preS1 (36-46), from 5 to 80 nM for preS1 (1-119), and from 100 to 800 lM for preS1 (1-11), preS1 (27)(28)(29)(30)(31)(32)(33)(34)(35), preS1 (66-76), and preS1 (95-104). Kinetic measurements were made and kinetic constants were derived with the BIAevaluation Version 3.0 software (BIAcore AB).…”

“…Of these, F35.25 is ad subtype-specific neutralizing antibody that recognizes preS1 [7]. On the other hand, MA 18/7 is ay subtype-specific neutralizing antibody that recognizes preS1 (30)(31)(32)(33)(34)(35) [8] and 5a-19-1 antibody recognizes ayw subtype preS1 (36)(37)(38)(39)(40)(41)(42)(43) [9].…”

Multiple hTAFII31-binding motifs in the intrinsically unfolded transcriptional activation domain of VP16

“…Given that the full-length VP16 TAD is intrinsically unstructured like p53 TAD or preS1 (4,24,25,29,30), we focused our efforts on delineating the location of pre-structured motifs within an unbound state of VP16 TAD and to examine their interactions with hTAFII31. VP16 TAD demonstrates an IUP where its backbone dynamics serves as a useful measure for detecting pre-structuring propensity even when pre-structuring tendency based upon NOE or CSI patterns is not evident.…”