A Frame-by-Frame Glance at Membrane Fusion Mechanisms: From Viral Infections to Fertilization.

Viral entry and fertilization are distinct biological processes that share a common mechanism: membrane fusion. In viral entry, enveloped viruses attach to the host cell membrane, triggering a series of conformational changes in the viral fusion proteins. This results in the exposure of a hydrophobic fusion peptide, which inserts into the host membrane and brings the viral and host membranes into close proximity. Subsequent structural rearrangements in opposing membranes lead to their fusion. Similarly, membrane fusion occurs when gametes merge during the fertilization process, though the exact mechanism remains unclear. Structural biology has played a pivotal role in elucidating the molecular mechanisms underlying membrane fusion. High-resolution structures of the viral and fertilization fusion-related proteins have provided valuable insights into the conformational changes that occur during this process. Understanding these mechanisms at a molecular level is essential for the development of antiviral therapeutics and tools to influence fertility. In this review, we will highlight the biological importance of membrane fusion and how protein structures have helped visualize both common elements and subtle divergences in the mechanisms behind fusion; in addition, we will examine the new tools that recent advances in structural biology provide researchers interested in a frame-by-frame understanding of membrane fusion.

Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation.

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcAG579R mutation abolishes this interaction. PkcAG579R also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity.

Solution structure of Plasmodium falciparum Hsp90 indicates a high flexible dimer.

Hsp90 is a ubiquitous, homodimer and modular molecular chaperone. Each Hsp90 protomer has three different domains, named the N-terminal domain (NTD), middle domain (MD) and C-terminal domain (CTD). The Hsp90 molecular cycle involves ATP binding and hydrolysis, which drive conformational changes. Hsp90 is critical for the viability of eukaryotic organisms, including the protozoan that causes the severe form of malaria, Plasmodium falciparum, the growth and differentiation of which are compromised when Hsp90 is inhibited. Here, we characterize the structure of a recombinant P. falciparum Hsp90 (PfHsp90) protein, as well as its MD (PfHsp90MD) and NTD plus MD (PfHsp90NMD) constructs. All the proteins were obtained with high purity and in the folded state. PfHsp90 and PfHsp90NMD interacted with adenosine nucleotides via the NTD, and Mg2+ was critical for strong binding. PfHsp90 behaved mostly as elongated and flexible dimers in solution, which dissociate with a sub-micromolar dissociation constant. The PfHsp90MD and PfHsp90NMD constructs behaved as globular and elongated monomers, respectively, confirming the importance of the CTD for dimerization. Small angle X-ray scattering data were obtained for all the constructs, and ab initio models were constructed, revealing PfHsp90 in an open conformation and as a greatly elongated and flexible protein.

Thermodynamic analysis of interactions of the Hsp90 with adenosine nucleotides: A comparative perspective.

Hsp90s are key proteins in cellular homeostasis since they interact with many client proteins. Several studies indicated that Hsp90s are potential targets for treating diseases, such as cancer or malaria. It has been shown that Hsp90s from different organisms have peculiarities despite their high sequence identity. Therefore, a detailed comparative analysis of several Hsp90 proteins is relevant to the overall understanding of their activity. Accordingly, the goal of this work was to evaluate the interaction of either ADP or ATP with recombinant Hsp90s from different organisms (human α and ß isoforms, Plasmodium falciparum, Leishmania braziliensis, yeast and sugarcane) by isothermal titration calorimetry. The measured thermodynamic signatures of those interactions indicated that despite the high identity among all Hsp90s, they have specific thermodynamic characteristics. Specifically, the interactions with ADP are driven by enthalpy but are opposed by entropy, whereas the interaction with ATP is driven by both enthalpy and entropy. Complimentary structural and molecular dynamics studies suggested that specific interactions with ADP that differ from those with ATP may contribute to the observed enthalpies and entropies. Altogether, the data suggest that selective inhibition may be more easily achieved using analogues of the Hsp90-ADP bound state than those of Hsp90-ATP bound state.

Comparative studies of the low-resolution structure of two p23 co-chaperones for Hsp90 identified in Plasmodium falciparum genome.

The p23 proteins are small acidic proteins that aid the functional cycle of the Hsp90 molecular chaperone. Such co-chaperone acts by temporarily inhibiting the ATPase activity of Hsp90 and exhibits intrinsic chaperone activity, suggesting independent roles. A search for p23 in the Plasmodium falciparum genome led to the identification of two putative proteins showing 13% identity to each other and approximately 20% identity to human p23. To understand the presence of two p23 proteins in this organism, we generated recombinant p23 proteins (Pfp23A and Pfp23B) and investigated their structure and function. The proteins presented some similarities and dissimilarities in structural contents and showed different chemical and thermal stabilities, with Pfp23A being more stable than Pfp23B, suggesting that these proteins may present different functions in this organism. Both Pfp23 proteins behaved as elongated monomers in solution and were capable of preventing the thermal-induced aggregation of model client proteins with different efficiencies. Finally, the Pfp23 proteins inhibited the ATPase activity of recombinant P. falciparum Hsp90 (PfHsp90). These results validate the studied proteins as p23 proteins and co-chaperones of PfHsp90.