Biology subjects

Muguruza-Montero, A.

Publications and source records attributed to Muguruza-Montero, A..

4 recordsLinked to original sources

bioRxiv2022

Molecular dynamics simulations of the calmodulin-induced alpha-helix in the SK2 calcium-gated potassium ion channel

The family of small-conductance (SK) ion channels is composed of four members (SK1, SK2, SK3, and SK4) involved in neuron-firing regulation. The gating of these channels depends on the intracellular Ca2+ concentration, and their sensitivity to this ion is provided by calmodulin (CaM). This protein binds to a specific region in SK channels known as the calmodulin-binding domain (CaMBD), an event which is essential for their gating. While CaM-binding domains are typically disordered in the absence of CaM, the SK2 channel subtype displays a small pre-folded -helical region in its CaMBD even if CaM is not present. This small helix is known to turn into a full -helix upon CaM binding, although the molecular-level details for this conversion are not fully understood yet. In this work, we offer new insights on this physiologically relevant process by means of enhanced sampling, atomistic Hamiltonian replica exchange molecular dynamics simulations, providing a more detailed understanding of CaM binding to this target. Our results show that CaM is necessary for inducing a full -helix along the SK2 CaMBD through hydrophobic interactions with V426 and L427. However, it is also necessary that W431 does not compete for these interactions; the role of the small pre-folded -helix in the SK2 CaMBD would be to stabilize W431 so that this is the case.

biophysics↗

bioRxiv2022

Improved KCNQ2 gene missense variant interpretation with artificial intelligence

Advances in DNA sequencing technologies have revolutionized rare disease diagnosis, resulting in an increasing volume of available genomic data. Despite this wealth of information and improved procedures to combine data from various sources, identifying the pathogenic causal variants and distinguishing between severe and benign variants remains a key challenge. Mutations in the Kv7.2 voltage-gated potassium channel gene (KCNQ2) have been linked to different subtypes of epilepsies, such as benign familial neonatal epilepsy (BFNE) and epileptic encephalopathy (EE). To date, there is a wide variety of genome-wide computational tools aiming at predicting the pathogenicity of variants. However, previous reports suggest that these genome-wide tools have limited applicability to the KCNQ2 gene related diseases due to overestimation of deleterious mutations and failure to correctly identify benign variants, being, therefore, of limited use in clinical practice. In this work, we found that combining readily available features, such as AlphaFold structural information, Missense Tolerance Ratio (MTR) and other commonly used protein descriptors, provides foundations to build reliable gene-specific machine learning ensemble models. Here, we present a transferable methodology able to accurately predict the pathogenicity of KCNQ2 missense variants with unprecedented sensitivity and specificity scores above 90%.

pathology↗

bioRxiv2022

Redox regulation of Kv7 channels through EF3 hand of calmodulin

Neuronal KV7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca2+-binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B. We found that precluding Ca2+ binding to the EF3 hand, but not to EF1, EF2 or EF4 hands, abolishes oxidation-induced enhancement of Kv7.4 currents. Monitoring FRET between helices A and B tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca2+, but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca2+ binding to EF1, EF2 or EF4 are negligible. Furthermore, we show that EF3 is necessary and sufficient to translate Ca2+ signals to reorient the AB fork. Our data is consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves Kv7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is necessary and sufficient for this signaling. SignificanceOxidation-dependent enhancement of the KV7/M-channels plays a cytoprotective role in neurons. Here, we show that calmodulin (CaM), the main protein that conveys information from transient intracellular Ca2+ oscillations, plays a critical role in oxidative signal transduction. The prevailing view is that the main role of the EF-hands is to respond to Ca2+ and that the two EF-hands of CaM in each lobe act in coordination during signaling. We find that EF3 by itself is sufficient and necessary for the oxidative response of Kv7 channel complex and for gating the Calcium Responsive Domain of Kv7 channels. In addition, the direction of EF3-dependent signaling can be reversed by protein-protein interactions with solvent exposed regions outside the target binding groove between EF-hands.

cell biology↗

bioRxiv2020

AN EPILEPSY-CAUSING MUTATION LEADS TO CO-TRANSLATIONAL MISFOLDING

Protein folding to the native state is particularly relevant in human diseases where inherited mutations lead to structural instability, aggregation and degradation. In general, the amino acid sequence carries all the necessary information for the native conformation, but the vectorial nature of translation can determine the folding outcome. Calmodulin (CaM) recognizes the properly folded Calcium Responsive Domain (CRD) of Kv7.2 channels. Within the IQ motif (helix A), the W344R mutation found in epileptic patients has negligible consequences for the structure of the complex as monitored by multiple in vitro binding assays and molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments.

biophysics↗