Guide Structure and Dynamics of Surfaces I

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  1. Product details
  2. Fusion surface structure, function, and dynamics of gamete fusogen HAP2
  3. Dynamics at Surfaces Conference GRC
  4. Nucleosome structure and dynamics are coming of age

Loops are labeled and 3-letter amino acid codes are used in the upper monomer and 1-letter codes are used in lower monomers. Helix axes in the right-most monomer are dashed. E Details of residues that support the structure of loops 1 and 2. G Detail of D2. H Orientations of D2. The division between fusion loops is defined by a basally projecting loop with Arg at its tip. The Arg sidechain guanido group docks through six hydrogen bonds to its carbonyl cage formed by backbone and sidechain carbonyl oxygens in the core of subdomain D2.

Thus, in the trimeric HAP2 state, the hydrophobic residues on the fusion helices in each monomer should also be able to interact with the lipid bilayer of the plus gamete. In the crystal structure, however, D2. Because of the large hydrophilic surface exposed on the faces of each monomer surrounding the 3-fold axis, deformation of lipids to fit into this large cavity, or the presence of water in the cavity, would limit the depth to which HAP2 trimers could insert in the hydrophobic core of the membrane bilayer during membrane fusion. These apparent barriers raise the possibility that our crystal structure represents an early fusion state of HAP2 that precedes a more mature state in which reorientation could allow the fusion loops in each monomer to approach and close up at the 3-fold axis to form a common fusion surface.

Comparisons among HAP2 and viral fusion state crystal structures and among monomers in these structures reveal motions that affect how closely fusion loops approach one another at the 3-fold axis Figures 5 and 6. While the three monomers in our HAP2 structure have essentially identical D2. Figure 5C—D compares three monomers from one of the two trimers in the Tick-borne encephalitis virus structure Bressanelli et al. Figure 5E—F compares monomers from the trimeric Dengue 1 and St. Louis encephalitis virus structures Luca et al. The most fusion loop-proximal residue that is conserved in position between HAP2 and flaviviral fusogens as shown by superposition of D2.

Thus, the HAP2 monomer in cyan and the Tick-borne encephalitis monomer in green differ from their counterpart monomers radially. In contrast, the HAP2 monomers in magenta and green and the Tick-borne encephaliti s monomers in cyan and magenta differ from one another in circumferential position Figure 5A—D. Among flavivirus fusion state structures, perhaps the largest difference in orientation at the D1-D2.

Louis encephalitis flavivirus fusogens Figure 5E,F and Figure 6.

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Viral class II fusogens also flex at the D1-D2. Flexibility visualized in comparison between flavivirus fusion state structures has both radial and circumferential components Figure 5E—F and is larger in extent than seen in the examples of D2. Flexibility at both junctions, D2. Bressanelli et al. Dengue 1 Nayak et al.

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Louis encephalitis Luca et al. Distances from a conserved D2. Dotted lines mark boundaries at which tilting occurs. Structures are shown in ribbon cartoon with disulfide bonds and key fusion loop sidechains in stick. Monomers are superimposed on D1. Additional marked differences in the fusion loops between the current and previous HAP2 structures are independent of D2.

Of the fusion loop segment from residue to , only fragments from residue to monomer A or from residue to or monomers B and C were built in the previous structure. Moreover, the entirety of these segments differs from that in our structure. The segment from Arg to Cys in the 2. The difference in conformation and the contraction might result from protease cleavage of the loops flanking this segment in the previous structure.

Whatever the cause, fusion loop residues Phe and Trp, present in only monomer A in the 3. Although the conformation of the fusion loop fragments in the previous structure differs from that in ours, these fragments are held in roughly the same position by binding of Arg to its carbonyl cage and disulfide linkage of Cys to Cys Arg and Cys are included in the shortest of the fragments previously traced, from residues to Thus, positioning of the Arg sidechain in the carbonyl cage of the D2.

HDX provided further evidence for differential mobility within the two fusion loops. Moreover, the backbone amide hydrogens in this fusion loop 1 peptide exchanged more rapidly in trimeric than monomeric HAP2 Figure 2D. These results are consistent with the paucity of backbone hydrogen bonds in the non-helical portion of this segment and its exposure to solvent in the trimeric structure. The kinetics of exchange in each overlapping peptide showed two distinct groups of residues, with one group of residues exchanging from 0 to 10 min, and another group not exchanging from 10 to min and thus highly stable Figure 2—figure supplement 2D-E.

Therefore, at least a portion of the loop two sequence W SDPLDIL , which contains three of five loop two residues implicated in fusion, has a stable structure in both the monomer and trimer. The increase in loop one and decrease in loop two dynamics suggest that both regions alter in structure or exposure upon trimer formation. In D2. These HDX results show that in most domains, regions in trimer interfaces are in more rapid exchange. Rapid exchange, which largely correlates with flexibility, may be a specialization that permits reshaping during monomer to trimer transition, and alterations in D2.

Our finding that the long fusion loop of HAP2 displayed three distinct helices, each projecting sets of hydrophobic residues that could interact with the lipid bilayer of the target membrane, was unexpected. This finding provided the opportunity to test the hypothesis that the residues in each helix had an equivalent functional effect on Chlamydomonas gamete fusion.

We examined the functional relevance of these hydrophobic residues by mutating them to Ala. HAP2 protein is expressed only when vegetatively growing minus cells are induced to become gametes. We first established proper surface localization in hap2 minus gametes.

Immunoblotting showed the typical HAP2 doublet with a larger surface-expressed form and a smaller, intracellular form Liu et al. HAP2 is localized in minus gametes to a small patch of membrane, the minus mating structure, between the two cilia. Anti-HA immunofluorescence combined with differential interference contrast DIC microscopy showed that wild-type and each mutant HAP2 were present between the two cilia at the site of the minus mating structure Figure 7C.

Thus, the three fusion helix mutations gave rise to HAP2 proteins that were of the correct size in SDS-PAGE, surface-expressed as shown by trypsin susceptibility, and localized to the mating structure as shown by microscopy. Blotting with anti-tubulin lower controlled for loading. C Combined immunofluorescence staining with anti-HA and differential interference contrast DIC microscopy show the location of wild type and HAP2 fusion helix mutant proteins on the minus mating structure between the two cilia arrow heads.

D Schematic illustration of steps left to right in Chlamydomonas fertilization. E-I Assays with the indicated mixtures of minus and plus gametes. E Ciliary adhesion at 5 min after mixing as assessed by particle counting. A Kruskal-Wallis test showed no significant difference in gamete activation among samples. G and H. Mating structure adhesion. G Quantification. Gamete fusion in Chlamydomonas is the culmination of a series of complex cellular events initiated when plus and minus gametes are mixed together Figure 7D.

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Successful gamete fusion, which occurs within minutes after gamete mixing, first requires that cells undergo ciliary adhesion that results in cellular agglutination. Ciliary adhesion-induced gamete activation then elicits release of cell walls and formation of membrane protuberances, the activated plus and minus mating structures.

Finally, the tips of these mating structures adhere to each other, followed by bilayer merger and complete cell coalescence to form a quadri-ciliated zygote. We established that expression of the fh HAP2 transgenes was without effect on these pre-fusion steps.

Fusion surface structure, function, and dynamics of gamete fusogen HAP2

Cellular agglutination as measured by electronic particle counting showed that fh minus gametes were indeed as capable of ciliary adhesion as wild-type minus gametes when mixed with plus gametes Figure 7E. In the next step of fertilization, plus and minus gametes lose their cell walls Figure 7D. Cell wall loss, as measured by the susceptibility of wall-less but not walled gametes to mild detergent-mediated release into the supernatant of cytoplasmic contents including ODdetectable chlorophyll, was robust and indistinguishable from wild-type in fh mutants Figure 7F , showing that they became activated.

All three fh mutants were competent in mating structure adhesion Figure 7G and H. Notably, mating structure adhesion, but not any earlier step, was abolished by the use of fus1 plus gametes, which lack an adhesion molecule required on plus gametes for mating structure adhesion Misamore et al. Finally, having established that HAP2 fh mutants were competent in all steps in fertilization leading up to fusion, we examined fusion itself.

Dynamics at Surfaces Conference GRC

Plus gametes were mixed with equal numbers of hap2 minus gametes expressing wild-type or fh mutant forms of HAP2-HA and fusion was assayed as the percent of gametes that had progressed from having two cilia to zygotes with four cilia Figure 7I. Fusion with fh2 gametes was significantly reduced and less than half of wild-type at all time points. These results demonstrate that the apically exposed, hydrophobic residues in each of the helices in the HAP2 fusion loops are indeed important in cell fusion during Chlamydomonas fertilization. Below, we interpret our structural and functional results, and structures of viral fusogens such as those shown in Figures 5 and 6 , in support of a model in which the fusion loops in each HAP2 monomer approach the 3-fold axis in the fusion state interrogated in vivo by mutation.

In this model, the HAP2 crystal structure represents an intermediate state in fusion.

Nucleosome structure and dynamics are coming of age

Pivoting or iris-like movements in D2. We have characterized the crystal structure of HAP2 from Chlamydomonas reinhardtii in a trimeric fusion state at 2. We were fortunate that our structure allowed us to completely trace the unusually long fusion loop in Chlamydomonas HAP2, revealing that the hydrophobic residues in its 2 loops are apically exposed on 3 helices.

Our structure also revealed other long loops in HAP2, which altogether make its sequence from D1 to D3 residues in total longer than in previously crystallized class II fusogens from viruses to residues or a C. It is possible that these long loops and glycans stabilize HAP2, or the fusing membrane bilayers, by decreasing the volume accessible to alternative disordered protein or membrane states. They may therefore have functional significance.

An alternative explanation is that the requirement for class II viral fusogens to pack tightly against one another and other proteins in an icosahedral lattice on viral surfaces may select against long loops and bulky glycans, whereas HAP2 should lack similar evolutionary constraints. HDX provided insights into the backbone dynamics of HAP2 and differences between its monomeric and trimeric states. Slower exchange at multiple domain-domain interfaces in the trimeric state compared to the monomeric state was consistent with less exposure or structural rearrangements upon trimer formation.

Regions of slower exchange in trimers included interfaces buried on D3, and buried on D1 and D2. Conversely, slower exchange in the monomeric state in a loop in D3 that is exposed in the trimeric state suggested that this loop could contact D1 in a more linear arrangement of domains in the monomer. Large HDX differences were also found in fusion loops 1 and 2 between the trimeric and monomeric forms, showing that these loops are capable of structural alterations or differ in exposure in these states.

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