Download The role of histidine residues in low-pH-mediated viral

The role of histidine
residues in
low-pH-mediated viral
membrane fusion
4
This chapter was adapted from the publications
Kampmann, T., D. S. Mueller, A. E. Mark, P. R. Young & B. Kobe. The role
of histidine residues in low-pH-mediated viral membrane fusion. Structure 14(10),
14811487 (2006)
and
Mueller, D. S., T. Kampmann, R. Yennamalli, P. R. Young, B. Kobe & A. E. Mark.
Histidine protonation and the activation of viral fusion proteins. Biochem. Soc. T.
36(Part 1), 4345 (2008).
In the article by Kampmann et al. [2006] I was responsible for the molecular dynamics
simulations of the dengue viral type 2 sE protein and wrote the corresponding parts of the
article. I also contributed substantially to the structural analyses of the sequence alignments
and the homology models. Among the studies reviewed in Mueller et al. [2008], written by
myself, I was responsible for the simulations of the wild type sE protein.
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4.1 Introduction
4.1
Introduction
Membrane fusion is an essential step during the entry of enveloped viruses into their host
cell. Depending on the type of virus, membrane fusion is initiated by various mechanisms,
including receptor binding (e.g. HIV-1), changes in pH (e.g. Influenza virus and Dengue
virus) or a combination of both (e.g. Avian sarcoma virus and Leukosis virus), with the
mechanism of fusion being related to the individual viral life cycle. Membrane fusion is a
fundamental biological process that occurs within a wide range of organisms and processes.
Often this process is promoted or catalysed by so-called fusion proteins that attach to the
membranes, thus prolonging the contact period and possibly enhancing interactions between
the membranes. Both viral and eukaryotic fusion proteins show a high degree of structural
similarity [Söllner 2004]. These findings raise the questions:
i) What are the underlying mechanisms that trigger fusion?
ii) Are these mechanisms common to one or more entire classes of viral fusion proteins?
Before the fusion event, viral surface proteins that drive fusion adopt a “meta-stable”
conformation, the so-called “pre-fusion” conformation. After a specific regulatory event
such as receptor binding or a change in pH, they undergo a series of structural transitions,
ultimately leading to the stable “post-fusion” conformation. All viral fusion proteins contain
a hydrophobic segment referred to as the “fusion peptide”, which is in most cases initially
buried within the pre-fusion form. However, once the conformational change is triggered,
the fusion peptide is exposed and can associate with the membrane of the host cell. In this
transition phase the protein is anchored simultaneously in the viral envelope and the host cell
membrane, and further conformational changes drive the two membranes to fuse [Harrison
2005; Schibli & Weissenhorn 2004].
Two major classes of viral membrane fusion proteins have been characterised:
Class I fusion proteins are found in the envelopes of viruses belonging to the Coronaviridae,
Filoviridae, Arenaviridae, Orthomyxoviridae, Paramyxoviridae and Retroviridae families [Colman & Lawrence 2003; Earp et al. 2004; Poumbourios et al. 1999]. The
influenza virus hemagglutinin (HA) is the archetypical class I fusion protein that has
been studied the most (Fig. 4.1A). In the pre-fusion conformation HA forms trimeric
spikes on the virion surface [Wilson et al. 1981]. During fusion the proteins rearrange,
forming post-fusion hairpin structures in which the C-terminal membrane anchor and
the fusion peptide are juxtaposed at the same end of a helical, rod-like structure [Bullough et al. 1994].
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4 Hypothesis: Histidine protonation triggers low-pH-mediated viral fusion
Class II fusion proteins such as those encoded by the viruses of the Togaviridae and Flaviviridae families have a very different molecular architecture than the class I proteins (Fig.
4.1) [Allison et al. 2001; Kielian 2006; Lee et al. 1997; Lescar et al. 2001; Modis et al.
2003; Rey et al. 1995]. They have three domains that are rich in β-structure, and their
fusion peptide is located within an internal loop. While the fusion proteins from different viruses in this class share a remarkably similar tertiary structure, the processing
of the fusion proteins differ. Flavivirus E, and alphavirus E1 fusion proteins initially
assemble as heterodimers, involving a companion viral surface protein that acts as a
folding chaperone, prM in flaviviruses, and PE2 in alphaviruses. Maturation of the
viral particle requires proteolytic cleavage of the chaperone by furin, leading to the rearrangement of the fusion protein as an E homodimer in flaviviruses, and as an E1/E2
heterodimer in alphaviruses, where E2 derives from PE2. Once activated the dimer
rearranges further into fusion protein homotrimers, which expose the fusion peptide at
the tip of a rod-like structure [Bressanelli et al. 2004; Gibbons et al. 2004; Modis et al.
2004].
Although the details of the conformational transitions in class I and class II proteins
differ, they nevertheless share common mechanistic features. Notably, in both cases the
protein folds back onto itself, causing the two membrane attachment points to be located
close together in the post-fusion conformation, which in turn facilitates membrane fusion
[Bressanelli et al. 2004; Bullough et al. 1994; Fass et al. 1996; Gibbons et al. 2004; Jardetzky
& Lamb 2004; Kielian & Rey 2006; Kobe et al. 1999; Modis et al. 2004; Roche et al. 2006;
Schibli & Weissenhorn 2004] (Figure 4.1).
Histidine is the only amino acid the protonation state of which changes near the pH of
fusion. In solution in water, histidine is predominantly uncharged at neutral pH, and doubly
protonated and positively charged at pH 6 and below. However, the effective pKa of a specific histidine ultimately depends on its local environment. Figure 4.2 illustrates the effect
of protein-internal interactions of histidine that significantly differ before and after histidine
protonation. In the pre-fusion state (left) the histidine residue is located in the vicinity of
positively charged residues (+ symbols). At low pH (middle) the histidine residue is doubly protonated. This will favour electrostatic interactions with negatively charged groups,
which may lead to the formation of new salt bridges (right) and thereby facilitate irreversible
refolding. Hydrogen bonds involving histidine as the acceptor will also be perturbed upon
protonation, due to repulsion from the donor. There are many examples of histidine protonation triggering structural changes at low pH [Nordlund et al. 2003], including changes
induced within the endosome [Lazar et al. 2003]. Therefore, histidine residues are expected
to play a critical role in the process of viral membrane fusion [Bressanelli et al. 2004; Chen
et al. 1998; Da Poian et al. 2005; Roussel et al. 2006; Stevens et al. 2004]. Questions re39
(left) and post-fusion (right) conformations. The ribbons represent the backbone conformation, the beads indicate selected residues. One subunit in
each protein complex is highlighted orange and cyan against the other subunits in grey. Colour legend: magenta histidines, blue positively charged,
red negatively charged; in A: orange HA1, cyan HA2; in B: orange domains I and II, cyan domain III.
Figure 4.1 The structures of A) the inuenza virus hemagglutinin (HA), and of B) the dengue viral envelope protein E, in the respective pre-fusion
4.1 Introduction
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4 Hypothesis: Histidine protonation triggers low-pH-mediated viral fusion
Figure 4.2 Schematic diagram of the interactions of a histidine residue in a viral fusion protein in
the pre-fusion and the post-fusion state.
garding the nature of this role remain, specifically:
iii) Are the initial steps in viral membrane fusion simply due to the general effects of an
increase in surface charge?
iv) Or is fusion triggered by the protonation of one or more critical histidine residues?
This chapter presents an extension of previous structural analyses [Bressanelli et al. 2004;
Chen et al. 1998; Roussel et al. 2006; Stevens et al. 2004], to consider the role that individual
histidine residues play, and whether these are similar in class I and class II viral fusion
proteins.
In sequence alignments of viral fusion proteins from class I and II, shown in Table 4.1, a
small number of highly conserved histidine residues were identified that lie in key structural
locations. Based on this it is proposed here that these specific histidine residues and their
interaction partners play a significant role in initiating the structural transition leading to
viral fusion, employing similar triggering mechanisms commonly involving :
1. the collocation of histidines and positively charged residues in the pre-fusion structure,
and
2. the protonation of these histidines, which promotes their rearrangement to form salt
bridges with specific negatively charged residues in the post-fusion structure.
In this chapter the experimental evidence for this hypothesis is reviewed.
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4.2 Activation of class I fusion proteins
4.2
Activation of class I fusion proteins
The prototypical class I fusion protein is the influenza virus protein HA. The X-ray crystal
structures of the three different forms of the protein, the precursor form, the pre-fusion form,
and a proteolytic fragment of the post-fusion form, have been determined [Bullough et al.
1994; Chen et al. 1998; Skehel & Wiley 2000; Wilson et al. 1981]. In the crystal structure of the pre-fusion form of HA, HA1 residue His184 and HA2 residues His106, His142
and His159 are located in the vicinity of positively charged residues; their locations within
the structure are highlighted in Figure 4.1A. Not only are these histidine residues themselves highly conserved among the 16 different influenza serotypes, but so are a number of
neighbouring residues in the pre-fusion structure. His142 forms salt bridges with Asp86 and
Asp90. In the post-fusion conformation both side chain nitrogen atoms of His106 may act
as donors in hydrogen bonds with neighbouring glutamic acid residues (Figure 4.1A). Although His106 is not strictly conserved, the sequence alignments shown in Table 4.1 suggest
that HA2 residue His111 may substitute for the role of His106 in some serotypes. Note that
in the crystal structures only fragments of the viral fusion proteins are resolved. In the crystal structure of the post-fusion form His184 of HA1 and His159 of HA2 were not resolved,
therefore their locations and environments are not known.
The role of histidines and their protonation may be viewed in two contexts:
1. In their neutral, singly protonated form, histidines interact strongly with the positively charged residues found in their vicinity in the pre-fusion form, usually through
hydrogen-bonds. This will effectively lock the structure into the pre-fusion form, until
the histidines become doubly-protonated. The proximity to positively charged residues
will make protonation more difficult by lowering the effective pKa value of the histidine and increasing the initial barrier to activation.
2. Once the histidines are doubly-protonated, their newly acquired charge will interact
with the environment and lead to the formation of new salt bridges with specific
negatively-charged residues. As illustrated in Figure 4.2, the formation of such salt
bridges will stabilise the doubly protonated form, resulting in an increase in the pKa of
the histidine, and possibly making the change effectively irreversible [Tanford 1970;
Warwicker 1989, 1992].
Previously it was proposed that histidine residues play important roles in the structural transitions of HA [Chen et al. 1998; Stevens et al. 2004]. An antigenic selection experiment of
HA identified His142 in HA2 [Nakajima et al. 2007] as a critical residue for the binding, and
a computational analysis identified regions surrounding several other histidines, as energetically critical for the overall stability of the protein [Isin et al. 2002]. In HA1 the mutation
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4 Hypothesis: Histidine protonation triggers low-pH-mediated viral fusion
of His17 to glutamine or arginine resulted in an increase in the pH of fusion [Daniels et al.
1985]. Note that whereas His17 is only partially conserved, it is located in the vicinity of
several other conserved histidines at positions 18 and 38 in HA1 and at 106 and 111 in HA2,
suggesting that these residues may regulate the structural transitions cooperatively.
4.3
Activation of class II fusion proteins
The prototypical class II fusion protein is the dengue virus E protein (Fig. 4.1B). In the
structure of the pre-fusion dimer [Modis et al. 2003], the highly conserved histidine residues
His244, His261 and His317 were located in the vicinity of positively charged residues, highlighted in Figure 4.1B. His261 and His317 form conserved salt bridges in the post-fusion
structure. His244 does not form a salt bridge in the post-fusion structure of the dengue viral E protein, but in the tick borne encephalitis (TBE) viral E protein the equivalent residue
His248 does form a salt bridge with Asp253. These histidine residues are located at molecular interfaces, His317 at the interface between domains I and III, and His244 and His261 at
the dimer interface — at interfaces which undergo extensive reorientation during conversion
to the post-fusion structure. As can be seen in the sequence alignments in Table 4.1B, the
residues that make up the local environment of these three histidines are also conserved in a
wide range of flaviviruses.
In the alphaviral Semliki Forest virus (SFV) envelope protein E1, residues His3 and
His125 also fit the pattern observed for the proposed key histidines in the fusion proteins
from influenza viruses and flaviviruses. It is possible that other histidines play similar roles
in fusion activation, but their identification has to wait until more complete structures become
available (e.g. the pre-fusion form of the E1-E2 complex of SFV). In SFV the substitution
His230Ala abrogated membrane fusion [Chanel-Vos & Kielian 2004]. While this residue is
structurally analogous to His244 in the dengue virus E protein, its role in fusion was not clear
from the available structures of the SFV E1 protein. Residues His146 and His323 in the TBE
virus, which were found to be equivalent to the respective residues His144 and His317 in the
dengue virus E protein, have been proposed as allowing the breakage of domain I/domain III
contacts at the initiation of the conformational changes leading to fusion [Bressanelli et al.
2004]. The environment of His144 in the dengue virus E protein is consistent with that
of other presumably important histidines discussed above; however, this residue formed a
hydrogen bond with a negatively charged residue already in the pre-fusion structure.
Critical interactions such as the salt bridge Arg9-Glu368 could be experimentally probed
in model systems through site-directed mutagenesis of the proposed residue partners, followed by examination of the fusion phenotype. The pre-fusion and the post-fusion structure
of the dengue virus E protein suggest that the Arg9–Glu368 salt bridge may act as a “linch43
For the legend and table B see next page.
Table 4.1 Sequence alignments of A) inuenza hemagglutinin HA, and B) aviviral envelope protein sE sequences.
4.3 Activation of class II fusion proteins
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aligned in their entirety, the table lists selected residues located within 0.5 nm of the side chain nitrogen atoms of selected histidines. A) Sequence
alignments of HA1 and HA2 sequences from 16 inuenza virus serotypes [Phipps et al. 2004]. Sequences for which an X-ray crystal structure has been
solved are indicated by their Protein Data Bank ID: 1HGJ, from the A/Aichi/2/1968 (H3N2) isolate [Sauter et al. 1992]; 1TI8, turkey H7 [Russell
et al. 2004]; 1JSM, avian H5 [Ha et al. 2002]; 1RVT, 1930-swine/human H1 [Gamblin et al. 2004]; 1JSD, swine H9 [Ha et al. 2002]. The last row
indicates the respective subunit, HA1 or HA2, of the heterodimer. B) Sequence alignment of twelve aviviral E protein sequences: from the four
dengue virus serotypes, DEN1, DEN2, DEN3 and DEN4, and closely related aviviruses, West Nile virus (WN), St. Louis encephalitis virus (SLE),
yellow fever virus (YF), Murray Valley encephalitis virus (MVE), Kunjin virus (KUN), tick-borne encephalitis virus (TBE), and Japanese encephalitis
virus (JE). X-ray crystal structures of E: 1OAN, DEN2 [Modis et al. 2003]; 1UZG, DEN3 [Modis et al. 2005]; 1SVB, TBE [Rey et al. 1995]. The last
row indicates the respective subunit, A or B, of the homodimer. Colour legend for residue types: magenta histidines; red negatively charged; blue
positively charged; orange polar; black non-polar; green aromatic.
Table 4.1 continued: Sequence alignments of A) inuenza hemagglutinin HA, and B) aviviral envelope protein sE sequences. The sequences were
4 Hypothesis: Histidine protonation triggers low-pH-mediated viral fusion
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4.4 Environment of histidine residues
pin”, maintaining the structure in the pre-fusion state.1 The conclusion is that once this
interaction is lost, the conformational transition from pre- to post-fusion state occurs spontaneously. It also has to be noted that the process is complex and aided by additional factors;
for example liposomes are required in addition to low pH for the transition of dengue and
TBE E protein ectodomains into the trimeric state [Modis et al. 2004; Stiasny et al. 2002]).
4.4 Environment of histidine residues
The propensity of specific histidines to be doubly protonated depends strongly on the local
environment, including the proximity of proton-donating groups and the degree of hydrophobic shielding from polar solvents [Tanford 1970; Warwicker 1989, 1992]. This provides a
means by which the protonation of critical histidines (and hence the triggering process) can
be modulated. While making protonation more difficult, the presence of positive charges
will provide a repulsive force driving conformational change, once the histidine side chain
is doubly protonated. An analogous model has been proposed for the mechanism of viral uncoating [Warwicker 1989, 1992]. The degree of accessibility to the solvent will also
modulate the effect of neighbouring positive charges on the pKa . In general, the less the
histidine side chain is accessible to the solvent, the stronger will be the effect of the protein
environment.
To determine whether the protonation of histidines could be the critical step in the structural transition from the pre- to the post-fusion conformation, molecular dynamics (MD)
simulations were performed of the pre-fusion dimer of the dengue virus sE protein. Two
systems were simulated, in which the histidine residues were either all singly- or all doublyprotonated, labelled HIS0 and HIS+ respectively. The results of these simulations are presented in Chapter 5. To further examine the specific roles of His244, His261 and His317,
simulations were performed in which combinations of these residues were selectively protonated Ragothaman & Kobe [2006–7]. To test the mechanical side-chain function of these
histidines, another series of simulations of a triple mutant was performed in which these histidine residues were mutated to alanine Ragothaman & Kobe [2006–7]. It was found that
when only a subset of histidines were either protonated or mutated to alanine, no significant
structural changes were observed within 20 ns of simulation. While this result does not indicate whether the specific residues identified can induce the conformational change, it was
most likely due to the limited timescale of the simulation.
1
linch-pin: “a pin passed through the end of an axle-tree to keep the wheel in its place” [Oxford English
Dictionary, Second Edition 1989].
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4 Hypothesis: Histidine protonation triggers low-pH-mediated viral fusion
4.5 Conclusion
The evidence presented in this chapter suggests that in a wide range of pH-activated viral
fusion proteins, the initial conformational changes associated with the transition from the
pre-fusion to the post-fusion form are triggered by the protonation of a small number of
conserved histidine residues. This conclusion is based on the analysis of structures of class I
and class II viral fusion proteins, and implies that in both classes the activation of the fusion
protein is triggered by an analogous molecular mechanism. Specifically, the model proposed
here stands in contrast to a general mechanism where conformational change is induced by
an increase in surface charge. In the pre-fusion form of the protein, the specific histidines are
located adjacent to positively charged residues. Protonation of these histidines would lead
to their expulsion and the subsequent formation of specific salt bridges, which could be the
reason that the transformation is essentially irreversible. The molecular surfaces involved
in the corresponding structural rearrangements leading to fusion are highly conserved and
might thus provide a suitable common target for the design of antivirals that could be active
against a diverse range of pathogenic viruses. The essential role of histidine residues may
even extend to proteins such as the glycoprotein G from Vesicular stomatitis virus, a member
of the Rhabdoviridae family, although the structural transition in this protein is reversible
[Carneiro et al. 2003]. The structure of the low-pH form determined very recently suggests
that upon deprotonation a number of residues including histidines would have a destabilising
effect [Roche et al. 2006].
As many of the viruses for which membrane fusion is induced at low pH are significant pathogens, the surfaces associated with the histidine-triggered structural rearrangements
might represent important potential target sites for antiviral compound design [Hoffman et al.
1997; Luo et al. 1997]. Indeed the conserved triggering mechanism of viral membrane fusion may open the possibility for the design of antiviral compounds with a broad spectrum
of efficacy.
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