Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

10C/2005G296

Interaction of calmodulin with the cytoplasmic domain of a transmembrane
gp41 : A novel binding mode of calmodulin molecular recognition
Yoshinobu IZUMI*, Akira AMANO, Takeshi SAITO, Yuji JINBO
Graduate School of Science & Eng., Yamagata Univ., Yonezawa, Yamagata 992-8510, JapanIntroduction
The HIV-1 envelope gp160 comprises a surface gp120
and a transmembrane gp41. Both are required for viralentry in to the cell. The gp41 is organized into threemajor regions: an N-terminal ectodomain, atransmembrane segment, and a C-terninal cytoplasmic tail.
Two calmodulin (CaM)-binding sites have been identified
near the cytoplasmic tail of gp41. Because the CaMbinding sites structurally and functionally resemble
natural cytolytic peptides, they are designated thelentivirus lytic peptides, LLP1 and LLP2.
Previous studies [1] reported that the potential to form ahighly positive charged amphipathic helix is essential forboth the membrane-perturbative and CaM-binding
activities of LLP1 and that CaM-binding function is wellreserved despite the sequence variation observed in nature.
However, it is still unknown about what mode ofmolecular recognition can be applied to the present CaMtarget recognition.
Experimental
The recombinant rat CaM was expressed and purified
as described previously [2]. Seven LLP1s were chosenfrom ENV_HV1A2 (aa 836-855; A2), HV1B1 (aa 837856; B1), and HV1H2 (aa 837-856: H2).
SR-SAXS has been employed to analyze the2+2+interaction of Ca /CaM with a LLP1. A Ca /CaM/RS20
complex was used as a standard sample.
Results2+
All ratios of the molecular weight of a Ca /CaM/
LLP1complex to the calculated value are almost in the2+neighborhood of 1.0, indicating that Ca /CaM binds each
peptide at the molar ratio of 1:1. The value of A2 for the2+
Ca /CaM/A2∆3 (∆ represents a truncation of amino acidresidues) complex is almost zero and that of Bif is negative,2+
while these values for Ca /CaM/A2∆10 complex are
positive, suggesting that the truncation at the C-terminus
increases the solubility of the complex, if the sequence istruncated at a basic amino acid residue. The R0 values for2+
the Ca /CaM/LLP1complexes take an intermediate value
between 21.6 A for a dumbbell-shaped structure and 18.0
A for a compact globular structure, suggesting that thesecomplexes adopt either a mixture of a dumbbell-shapedstructure and a globular structure or a novel structure.
The data points in the Kratky plots are almost
2+
superimposed to those for the Ca /CaM/RS20 complex,
whose complex adopts a compact globular shape [3].
Discussion
The results for the SAXS measurements presented here
show that Ca2+/CaM binds each LLP1 at a molar ratio of
1:1, each complex adopts almost the same globularstructure as that for the Ca2+/CaM/RS20 complex,
suggesting that each LLP1 adopts an alpha-helicalstructure in the complex.
As the complex with
HV1A2∆3 adopts almost the same globular structure asthat with HV1A2∆10, the CaM-binding site is containedin the latter sequence. However, no CaM-binding modein this sequence is retrieved using the CaM target
database [4]. Moreover, Ca2+/CaM binds a peptide withthe opposite sequence and adopts almost the same
globular structure as that for the Ca2+/CaM/RS20complex. At the two opposite outlets of the hydrophobicchannel composed by two domains of CaM are clusters ofacidic residues that are asymmetric in size [5]. TheLLP1s that interacts with this channel possess
complementary polarity created by a cluster of basic
residues in the amino acid sequences. The basic clusterof RS20 is located at the N-terminal side of the CaMbinding region, while that of the LLP1s is located at theC-terminal side, as shown in Fig.1. Taken together, theresults indicate that the location of a basic cluster within
the CaM-binding region plays an important role indetermining the direction of its binding with respect toCaM domains [5]. Furthermore, the binding mode ofCa2+/CaM molecular recognition is well preserved despiteof the sequence variation of three species of gp41 asshown in Fig. 1, suggesting that this region of theglycoprotein is important to viral replication.
NCNC

HV1A2∆10:DRVIEVAQRAYRAILHIHRR
HV1B1∆10:DRVIEVVQGAYRAIRHIPRR
HV1H2∆10:DRVIEVVQGACRAIRHIPRR
2+Fig. 1. Schematic model of Ca /CaM/LLP1 complex:
2+N and C-terminal domains of Ca /CaM are shown in
red and orange, respectively. N and C-terminal halvesof the target peptide are shown in cyan and blue,respectively.
References[1] S.B.Tencza et al., J.Virol.69, 5199(1995).
[2] N.Hayashi et al., Protein Expr.Purif. 12, 25(1998).
[3] W.E.Meador et al., Science 257, 1251(1992).
[4] K.L.Yap et al., J. Struct. & Func. Geno. 1, 8(2000).
[5] M.Osawa et al., Nat. Struc. Biol. 6, 819(1999).
* yizimi@yz.yamagata-u.ac.jp

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