MEMBRANE BINDING AND OLIGOMERIZATION OF EIAV MATRIX PROTEIN

1
Abstract
HIV (human immunodeficiency virus) and EIAV
(equine infectious anemia virus) are closely
related lentiviruses that both infect immune
cells but whose pathogenesis differs. Membrane
binding of the (matrix) MA protein of HIV appears
to be primarily driven by a cluster of basic
residues in the MA domain and possibly assisted
by an N-myristoylation signal. Interestingly,
the MA protein of EIAV does not contain either of
these signals. To understand what factors may
promote EIAV assembly we characterized the
membrane binding properties of its MA proteins
using fluorescence methods and compared them to
our previous HIV-MA results. We find that like
HIV-MA, EIAV-MA exists as a multimer in solution
whose protein-protein interactions are
destabilized by membrane binding. Unlike HIV-MA,
EIAV-MA binds strongly to electrically neutral
membranes (POPC) as well as negatively charged
(POPS) ones and our results indicate a different
exposure of the EIAV-MA Trp residues when bound
to the two types of membranes. Based on these
data and the known structures of closely related
matrix proteins, we constructed a structural
model of EIAV-MA. This model predicts that
EIAV-MA binds to POPS similar to HIV-MA, but
EIAV-MA has an additional membrane binding region
that allows for hydrophobic membrane interactions.
2
Figure 1 Gene map of Gag and depiction of mature
virus assembly Matrix protein is instrumental in
membrane binding of Gag
3
-Decrease in fluorescence EIAV homotransfer as
seen by an increase in anisotropy as the pH is
lowered -Decrease in homotransfer indicates
subunit dissociation -This was verified by
SDS-PAGE electrophoresis (data not shown)
Figure 2 pH dependence of oligomerization.
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Figure 3 Membrane Binding of EIAV -Binding to
LUVs was followed by the decrease in intrinsic
fluorescence of EIAV-MA -Lipid membrane was added
to a solution containing EIAV-MA. This may
promote protein-protein interactions so an
alternate assay was done (Fig. 4)
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Figure 4 Membrane Binding of EIAV-MA to POPS and
POPC - Membranes were labeled with the
environmentally-sensitive probe Laurdan. The
shift in Laurdan fluorescence as the protein
displaces water from the surface was followed and
is shown.
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Figure 5 Quenching of Trp by KI differs when
EIAV is bound to POPS and POPC
80 uM POPS slope 0.0056
80 uM POPC slope 0.0039
7
Results

• Oligomerization of EIAV-MA is pH dependent as
  dissociation occurs at a pH of 4.5 (Fig. 2)
• EIAV-MA shows strong binding to lipid membranes
  regardless of membrane composition (A), salt
  concentration (B), or pH (C). (Fig. 3 and Fig. 4)
• Trp quenching by KI is an indication of where the
  hydrophobic residues are oriented after membrane
  binding. The observed difference between POPC
  and POPS indicates that EIAV-MA is binding
  differently to the two membranes. (Fig. 5)

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Figure 6 Molecular model for the EIAV-MA
structure. The amino acid sequences of the EIAV
and HIV-1 matrix domains were aligned with the
multiple sequence alignment program ClustalW
(Higgins et al., 1991). The sequence identity of
the alignment is 19 (23 identities over 120
residues). Homology models of the EIAV matrix
protein in monomeric and trimeric forms were
constructed with the model routine of the
homology modeling program Modeller (Sali and
Blundell, 1993). The ClustalW alignment was used
to match the EIAV matrix sequence to the HIV-1
matrix sequence the NMR structure of HIV-1
matrix protein (PDB code, 1tam) was used as the
structural template for the monomer model and the
x-ray structure of the HIV-1 matrix protein
trimer (PDB code, 1hiw) was used as the template
for the trimer model. Residues present in the
HIV-1 MA trimer interfaces are only partly
conserved in EIAV MA HIV-1 MA trimer interface
residues ERFAVNQQQTGS-EE Corresponding residues
in EIAV MA DLFHDTDLQTLSGEE Models of the EIAV
monomer based on other alignments share common
properties with the model described above 1)
electrostatic polarity the front surface is
basic, while the back surface is slightly
acidic with exposed hydrophobic residues that may
penetrate the membrane interface 2) surface Trp
residues are more prominent on the hydrophobic
face than the basic face 3) the sole Cys residue
is partially buried in the hydrophobic core.
Fluorescence studies measuring the accessibility
of this Cys support this model.
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Figure 7 Theoretical model of Membrane Binding
of EIAV-MA to POPS and POPC From the binding data
presented here we theorize that EIAV-MA binds to
POPS through electrostatic interactions. The
binding then promotes subunit dissociation
through the use of the anionic phosphates of the
membrane surface. We believe that EIAV-MA binds
to POPC and the subunits dissociate through the
same mechanisms as POPS. However, the
dissociation exposes hydrophobic residues found
within the slightly acidic region on the back
of the protein, and rolling of the protein for
the hydrophobic residues to penetrate the
membrane occurs on POPC.
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Conclusions

• Similar to HIV-MA
• The EIAV-MA solution structure is an oligomer
  that dissociates upon membrane binding.
• The penetration into the membrane surface is
  negligible.
• Unlike HIV-MA
• EIAV-MA has neither a myristoylation signal nor
  an apparent cluster of basic residues.
• The oligomerization of EIAV-MA is pH-dependent
  and the protein must use the anionic phosphates
  of the membrane surface to induce dissociation.
• EIAV-MA binds to electrically neutral membranes,
  however this binding may occur by alternate
  interaction sites that change the accessibility
  of its Trp residues. Energy transfer from EIAV-MA
  Trp to membrane-incorporated anthroyl stearic
  acid acceptors support this idea. Biochemical
  studies are now underway to test this model.

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Reference List
1. Higgins, D.G., Bleasby, A.J., and Fuchs, R.
(1991). ClustalW improved software for
multiple sequence alignment. CABIOS 8,
189-191. 2. Sali, A. and Blundell, T.J. (1993).
Comparative protein modeling by satisfaction
of spatial restraints. J.Molec.Biol. 234,
779-815. 3. Scarlata, S., Ehrlich, L.S., and
Carter, C.A. (1998). Membrane-induced
alterations in HIV-1 Gag and matrix
protein-protein interactions. J.Mol.Biol.
277, 161-169. 4. Ehrlich, L.S., Fong, S.,
Scarlata, S., Zybarth, G., and Carter, C.
(1996). Partitioning of HIV-1 Gag and
Gag-related proteins to membranes.
Biochemistry. 35, 3933-3943.
Acknowledgements
N. Tijandra, I. Jayatilaka Supported by NIH
05827101