Karina Alleva

Intracellular pH regulation of PIP2 aquaporins, primarily mediated by a histidine sensor in loop D, is essential for controlling water transport. While the C-terminal (CT) domain is involved, the precise contribution of the N-terminal (NT) and other flexible regions has been debated. Given that PIPs are small proteins that lack large regulatory domains yet achieve finely tuned gating, we sought to investigate how local structural flexibility and inter-region communication contribute to this process. To this end, we introduced alanine substitutions into conserved proline motifs in BvPIP2;2, targeting a contiguous NT PPP motif (BvPIP2;2-AAA) and two isolated prolines in loop D (BvPIP2;2-P194A and BvPIP2;2-P204A). Using heterologous expression in Xenopus laevis oocytes, we evaluated channel function and localization, while the structural impact of the mutations was analyzed with molecular dynamics (MD) simulations on full-length structural models. While BvPIP2;2-P204A impaired membrane trafficking, both BvPIP2;2-AAA and BvPIP2;2-P194A mutants localized to the plasma membrane and remained functional. Functional assays revealed that the BvPIP2;2-AAA mutant exhibited a marked alkaline shift in pH0.5, greater than that observed for BvPIP2;2-P194A. MD simulations showed that the NT mutation reduced flexibility, reorganized the network of interactions between NT and CT regions, and altered the environment and the pKa of the canonical pH sensor, His202. In contrast, the P194A mutation produced more modest structural and functional changes. These findings support a model in which transitions in structural flexibility within intracellular regions, particularly the NT region, govern communication between dynamic segments and modulate the electrostatic environment of the pH sensor, thereby fine-tuning pH gating in plant aquaporins.

Hydrogen peroxide (H₂O₂) is a key reactive oxygen species involved in cellular redox signaling and oxidative stress. Due to its polar nature, its transport across membranes is regulated by aquaporins (AQPs), membrane channels traditionally known for H₂O transport. Certain AQPs, known as peroxiporins, facilitate selective H₂O₂ permeation, playing critical roles in mantaining redox homeostasis. This review summarizes insights from molecular dynamics (MD) simulations into the mechanisms of H₂O₂ transport through AQPs. Key structural regions, such as the selectivity filter (SF) and NPA motif, influence H₂O₂ permeation, with energy profiles revealing differences from H₂O transport. While molecular mimicry suggests similarities in the movement of H₂O and H₂O₂, specific interactions and energetic barriers highlight the complexity of the process. We highlight the need for integrating computational and experimental findings for further studies to unify mechanistic understanding and develop applications in redox biology.

Hydrogen peroxide (H2O2) transport by aquaporins (AQP) is a critical feature for cellular redox signaling. However, the H2O2 permeation mechanism through these channels remains poorly understood. Through functional assays, two Plasma membrane Intrinsic Protein (PIP) AQP from Medicago truncatula, MtPIP2;2 and MtPIP2;3 have been identified as pH-gated channels capable of facilitating the permeation of both water (H2O) and H2O2. Employing a combination of unbiased and enhanced sampling molecular dynamics simulations, we investigated the key barriers and translocation mechanisms governing H2O2 permeation through these AQP in both open and closed conformational states. Our findings reveal that both H2O and H2O2 encounter their primary permeation barrier within the selectivity filter (SF) region of MtPIP2;3. In addition to the SF barrier, a second energetic barrier at the NPA (asparagine-proline-alanine) region that is more restrictive for the passage of H2O2 than for H2O, was found. This behavior can be attributed to a dissimilar geometric arrangement and hydrogen bonding profile between both molecules in this area. Collectively, these findings suggest mechanistic heterogeneity in H2O and H2O2 permeation through PIPs.

Interaction between membrane proteins and ligands plays a key role in governing a wide spectrum of cellular processes. These interactions can provide a cooperative-type regulation of protein function. A wide variety of proteins, including enzymes, channels, transporters, and receptors, displays cooperative behavior in their interactions with ligands. Moreover, the ligands involved encompass a vast diversity and include specific molecules or ions that bind to specific binding sites. In this review, our particular focus is on the interaction between integral membrane proteins and ligands that can present multiple “binding sites”, such as protons or membrane phospholipids. The study of the interaction that protons or lipids have with membrane proteins often presents challenges for classical mechanistic modeling approaches. In this regard, we show that, like Hill’s pioneering work on hemoglobin regulation, phenomenological modeling constitutes a powerful tool for capturing essential features of these systems.

Interaction between membrane proteins and ligands is key to control a wide spectrum of cellular processes. These interactions can provide a cooperative-type regulation of protein function. A wide diversity of proteins, including enzymes, channels, transporters, and receptors show cooperative behavior. The variety of possible ligands is also huge and includes specific molecules or ions that bind to an also specific binding site. In this review, we focus on the interaction of membrane proteins with ligands that present many binding sites, such as protons or membrane phospholipids. The study of the interaction that protons or lipids have with membrane proteins does not always allow a clear and classic mechanistic modeling approach. In this regard we show that, like Hill´s pioneering work on hemoglobin regulation, phenomenological modeling constitutes a powerful tool to describe relevant features of these important systems.

α-hemolysin (HlyA) of E. coli binds irreversibly to human erythrocytes and induces cell swelling, ultimately leading to hemolysis. We characterized the mechanism involved in water transport induced by HlyA and analyzed how swelling and hemolysis might be coupled. Osmotic water permeability (P_f) was assessed by stopped-flow light scattering. Preincubation with HlyA strongly reduced P_f in control- and aquaporin 1-null red blood cells, although the relative P_f decrease was similar in both cell types. The dynamics of cell volume and hemolysis on RBCs was assessed by electrical impedance, light dispersion and hemoglobin release. Results show that HlyA induced erythrocyte swelling, which is enhanced by purinergic signaling, and is coupled to osmotic hemolysis. We propose a mathematical model of HlyA activity where the kinetics of cell volume and hemolysis in human erythrocytes depend on the flux of osmolytes across the membrane, and on the maximum volume that these cells can tolerate. Our results provide new insights for understanding signaling and cytotoxicity mediated by HlyA in erythrocytes.

Major Intrinsic Proteins (MIPs) are membrane channels that permeate water and other small solutes. Some trypanosomatid MIPs mediate the uptake of antiparasitic compounds, placing them as potential drug targets. However, a thorough study of the diversity of these channels is still missing. Here we place trypanosomatid channels in the sequence-function space of the large MIP superfamily through a sequence similarity network. This analysis exposes that trypanosomatid aquaporins integrate a distant cluster from the currently defined MIP families, here named aquaporin X (AQPX). Our phylogenetic analyses reveal that trypanosomatid MIPs distribute exclusively between aquaglyceroporin (GLP) and AQPX, being the AQPX family expanded in the Metakinetoplastina common ancestor before the origin of the parasitic order Trypanosomatida. Synteny analysis shows how African trypanosomes specifically lost AQPXs, whereas American trypanosomes specifically lost GLPs. AQPXs diverge from already described MIPs on crucial residues. Together, our results expose the diversity of trypanosomatid MIPs and will aid further functional, structural, and physiological research needed to face the potentiality of the AQPXs as gateways for trypanocidal drugs.

Plant PIP aquaporins play a central role in controlling plant water status. The current structural model for PIP pH‐gating states that the main pH sensor is located in loopD and that all the mobile cytosolic elements participate in a complex interaction network that ensures the closed structure. However, the precise participation of the last part of the C‐terminal domain (CT) in PIP pH gating remains unknown. This last part has not been resolved in PIP crystal structures and is a key difference between PIP1 and PIP2 paralogues. Here, by a combined experimental and computational approach, we provide data about the role of CT in pH gating of Beta vulgaris PIP. We demonstrate that the length of CT and the positive charge located among its last residues modulate the pH at which the open/closed transition occurs. We also postulate a molecular‐based mechanism for the differential pH sensing in PIP homo‐ or heterotetramers by performing atomistic molecular dynamics simulations (MDS) on complete models of PIP tetramers. Our findings show that the last part of CT can affect the environment of loopD pH sensors in the closed state. Results presented herein contribute to the understanding of how the characteristics of CT in PIP channels play a crucial role in determining the pH at which water transport through these channels is blocked, highlighting the relevance of the differentially conserved very last residues in PIP1 and PIP2 paralogues.

Summary: Plasma membrane (PM) intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and small solutes. The functional importance of the PM organisation of PIPs in the interaction with other cellular structures is not completely understood. We performed a pull‐down assay using maize (Zea mays) suspension cells expressing YFP‐ZmPIP2;5 and validated the protein interactions by yeast split‐ubiquitin and bimolecular fluorescence complementation assays. We expressed interacting proteins tagged with fluorescent proteins in Nicotiana benthamiana leaves and performed water transport assays in oocytes. Finally, a phylogenetic analysis was conducted. The PM‐located ZmPIP2;5 physically interacts with the endoplasmic reticulum (ER) resident ZmVAP27‐1. This interaction requires the ZmVAP27‐1 cytoplasmic major sperm domain. ZmPIP2;5 and ZmVAP27‐1 localise in close vicinity in ER–PM contact sites (EPCSs) and endocytic structures upon exposure to salt stress conditions. This interaction enhances PM water permeability in oocytes. Similarly, the Arabidopsis ZmVAP27‐1 paralogue, AtVAP27‐1, interacts with the AtPIP2;7 aquaporin. Together, these data indicate that the PIP2–VAP27 interaction in EPCSs is evolutionarily conserved, and suggest that VAP27 might stabilise the aquaporins and guide their endocytosis in response to salt stress.