A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur at pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformational change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.Viruses such as influenza that contain lipid membranes appear to enter cells during infection by a process involving the fusion of the viral membrane with a cellular membrane. The results of recent investigations have indicated that, for a number of viruses, fusion occurs optimally over narrow ranges of pH, in the case of influenza viruses between pH 5.0 and pH 5.5 (1-4), and it has been proposed that this correlates with the pH at the site of cell entry in intracellular vesicles such as lysosomes (5).Evidence that the hemagglutinin (HA) glycoprotein is involved in influenza virus-mediated fusion includes the observations that post-translational cleavage ofa precursor HA, HAO, to HA1 and HA2 is required for both virus infectivity (6, 7) and in vitro virus-mediated fusion (4,8) and that the hydrophobic amino-terminal sequence of HA2 is analogous to that of the amino terminus ofthe F1 component of Sendai virus fusion glycoprotein (9-11). Furthermore, the findings that the amino-terminal sequence of HA2 consists of 10 uncharged hydrophobic amino acids (9) and is the most highly conserved sequence in the hemagglutinin (12) suggest that the terminal region may be directly involved in the membrane-fusion reaction. Analysis of the three-dimensional structure (12) of bromelain-released HA (BHA), which lacks the carboxyl-terminal hydrophobic region through which the complete HA is associated with the lipid membrane of the virus particle, suggests that a conformational .change may be required before this can occur.In this investigation, a conformational change of BHA from X-31 (H3N2) influenza virus has been observed, which occurs at pH values corresponding to those optimal 'for the in vitro membrane-fusion activity of the virus. CD, sedimentation, electron microscopy, and proteolytic susceptibility studies have been. used to characterize the pH 5.0-induced transition. The experiments lead to the conclusion that, after incubation at low pH, BHA can form hydrophobic associations with other BHA molecules, with lipid vesicles, or with nonionic detergent micelles. These results are discussed with reference to the threedimensional structure of HA and in relation to its possible role in virus-mediated fusion. METHODSVirus and HA Purification. X-31 (H3N2) influenza virus (13) was grown in embryonated hens' e...

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WFlO (residues 1-10 of M13) binds to CaM with Kd = 1 pM; peptide FWlO (residues 9-18 of M13, with Phe-17 -+ Trp substitution) binds to CaM with Kd = 100 pM. The effect of peptide WFlO on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable halfsaturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WFlO from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N-and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca 2ϩ or Mg 2ϩ . The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low DG8 20 values of ;2.7~N-domain! and ;1.9 kcal0mol~C-domain!. The apo-C-domain is significantly unfolded at normal temperatures~20-25 8C!. The greater affinity of the C-domain for Ca 2ϩ causes it to be more stable than the N-domain at @Ca 2ϩ # Ն0.3 mM. By contrast, Mg 2ϩ causes a greater stabilization of the N-rather than the C-domain, consistent with measured Mg 2ϩ affinities. For the intact protein~6Ca 2ϩ !, the bimodal denaturation profiles can be analyzed to give two DG8 20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.

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The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.

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Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.