A large‐scale evolutionary and structural analysis of CLC channels and transporters

“A linear morph between the two models was created and visualized using visual molecular dynamics (VMD) (Humphrey et al., 1996).”

“However, recent advances in sequence-to-structure prediction with AlphaFold2 (AF2) introduce a potential new source of information that can bridge this gap (Jumper et al., 2021).”

“Heat-maps of DMs and DDMs are made using the Python visualization library Matplotlib (Hunter, 2007).”

“The final tree was constructed with RAxML (LG + G model, 100 bootstrap replicates) and visualized with iToL (Letunic & Bork, 2021).”

“RFCs were trained using the Python machine-learning library Scikit-Learn (Pedregosa et al., 2011) on selected transporter and channel structures with the following parameters: n_estimators = 500, max_ depth = 5, max_samples = 0.3.”

“Escherichia coli have three CLC genes: CLC-ec1, CLC-ec2, and the uncharacterized ecYfeO (Blattner et al., 1997; Iyer et al., 2002).”

“We tested different sequence alignment strategies by constructing approximate trees with FastTree (same settings as before) on all eukaryotic CLC homologs.”

“The final tree was constructed with RAxML (LG + G model, 100 bootstrap replicates) and visualized with iToL (Letunic & Bork, 2021).”

“The first published structures were of CLC-st1 and CLC-ec1 (Dutzler et al., 2002) (Figure 1a), showing that each subunit is formed by 18 helices, α A-R, of which 16 are fully membrane embedded, α B-Q (Figure 1b).”

“At the onset of this study, there were nearly 75 K CLC sequences identified in the InterPro database (Blum et al., 2025) but only 13 genes represented in 106 experimentally solved structures, 51 of which are mutants (Table S1, Supporting Information).”

“These proteins play vital physiological roles governing the excitability of muscle and neurons (Bennetts et al., 2005; Guzman et al., 2014), acidification of lysosomes and bones (Kornak et al., 2001; Zifarelli, 2022), and acid resistance in certain bacteria (Gut et al., 2006).”

“In CLC-ec1, chloride ions are coordinated by residues S107 (helix α D) and Y445 (helix α R) at the central binding site, with E148 (helix α F) serving as a gate for the external site (Dutzler et al., 2003).”

“CLC-ec1, like all the other prokaryotic CLCs whose structures have been solved so far, functions as a secondary active transporter (Accardi & Miller, 2004).”

“This process was repeated for all partitions, providing a robust measure of classifier accuracy (Bates et al., 2024).”

“Although this study involved pairwise alignments of homologs with CLC-ec1 using TM-align, the multiple-structural alignment program US-align (C. Zhang et al., 2022) is an alternate method that may be useful for accelerating future analyses.”

“The others, hCLC-3, hCLC-4, hCLC-5, hCLC-6, and hCLC-7 have been demonstrated to act as coupled chloride/proton antiporters (Guzman et al., 2013; Leisle et al., 2011; Neagoe et al., 2010; Picollo & Pusch, 2005; Scheel et al., 2005).”

“…et al., 2005), and a putative channel ngCLC (Fortea Verdejo, 2021); transporter homologs include hCLC-3 (Guzman et al., 2013), hCLC-4, hCLC-5 (Picollo & Pusch, 2005; Scheel et al., 2005), hCLC-6 (Neagoe et al., 2010), hCLC-7 (Leisle et al., 2011), atCLC-a (De Angeli et al., 2006), and cmCLC…”

“Further, archaeal sequences are scattered throughout the tree, a possible result of either very early horizontal gene transfer (Boto, 2009) or low phylogenetic signal.”

“Common to many transporters (Pornillos & Chang, 2006), CLC adopts an inverted-topology domain repeat with helices α B-I forming the first half, and α J-Q forming the second (Forrest & Rudnick, 2009).”

“…this switch occurred, denoted the transition region, was defined as a low-resolution region of the tree containing both channel and transporter homologs, including transporter cmCLC (Feng et al., 2010) and channel ngCLC (Fortea Verdejo, 2021), located between two nodes with 96% support (Figure 2d).”

“Channel homologs include CLC-0 (Lísal & Maduke, 2008), hCLC-Ka/b (Kieferle et al., 1994), hCLC-1 (Saviane et al., 1999), hCLC-2 (Britton et al., 2005), and a putative channel ngCLC (Fortea Verdejo, 2021); transporter homologs include hCLC-3 (Guzman et al., 2013), hCLC-4, hCLC-5 (Picollo & Pusch,…”

“However, this limitation may be addressable as studies show that AF2 can be guided to sample alternative conformations through reducing input multiple sequence alignment (MSA) depth (del Alamo et al., 2022) or directly changing residues at certain alignment sites of the input MSA (Stein & Mchaourab, 2022).”

“The others, hCLC-3, hCLC-4, hCLC-5, hCLC-6, and hCLC-7 have been demonstrated to act as coupled chloride/proton antiporters (Guzman et al., 2013; Leisle et al., 2011; Neagoe et al., 2010; Picollo & Pusch, 2005; Scheel et al., 2005).”

“Escherichia coli have three CLC genes: CLC-ec1, CLC-ec2, and the uncharacterized ecYfeO (Blattner et al., 1997; Iyer et al., 2002).”

“Although AF2 tends to predict similar proteins in overly similar conformations (Saldaño et al., 2022), the large number of predicted structures is expected to provide a reasonable sampling of the structure space.”

“Channel homologs include CLC-0 (Lísal & Maduke, 2008), hCLC-Ka/b (Kieferle et al., 1994), hCLC-1 (Saviane et al., 1999), hCLC-2 (Britton et al., 2005), and a putative channel ngCLC (Fortea Verdejo, 2021); transporter homologs include hCLC-3 (Guzman et al., 2013), hCLC-4, hCLC-5 (Picollo & Pusch,…”

“Indeed, AF2 has been shown to be less effective at predicting the structure of fold-switching proteins (Chakravarty & Porter, 2022), revealing its inherent bias toward structures in the training set.”

“To identify how our classifier makes its predictions, we employed SHAP (SHapley Additive exPlanations) values, a method that quantifies the expected marginal contribution of each input feature to the classifier ’ s output for a single prediction (Lundberg & Lee, 2017; Winter, 2002).”

“…structural differences between any two CLCs regardless of sequence similarity, we calculate a DM aln for each structure and calculate the absolute value of its difference to obtain a DDM. High values in the DDM indicate structural changes between aligned residue pairs (Nishikawa et al., 1972).”

“These proteins play vital physiological roles governing the excitability of muscle and neurons (Bennetts et al., 2005; Guzman et al., 2014), acidification of lysosomes and bones (Kornak et al., 2001; Zifarelli, 2022), and acid resistance in certain bacteria (Gut et al., 2006).”

“Miller, 2007; Robertson et al., 2010).”

“…human homologs, hCLC-1, hCLC-2, hCLC-Ka, and hCLC-Kb have been shown to function as channels (Stölting et al., 2014), identified by an accelerated rate of anion permeation and the absence of proton transport linked to chloride permeation as indicated by reversal potential (Accardi & Picollo, 2010).”

“On the other hand, mutating the external glutamate and central binding site tyrosine (E148 and Y445) to smaller side-chains creates a wide aqueous pathway across the membrane (Jayaram et al., 2008), increasing the rate of chloride transport and effectively creating a channelized transporter hybrid.”

“These proteins play vital physiological roles governing the excitability of muscle and neurons (Bennetts et al., 2005; Guzman et al., 2014), acidification of lysosomes and bones (Kornak et al., 2001; Zifarelli, 2022), and acid resistance in certain bacteria (Gut et al., 2006).”

“Humans have nine different versions of CLCs demonstrating a variety of functions; mutations in these proteins lead to diseases such as Bartter ’ s syndrome or muscle myotonia (Stölting et al., 2014).”

“Channel homologs include CLC-0 (Lísal & Maduke, 2008), hCLC-Ka/b (Kieferle et al., 1994), hCLC-1 (Saviane et al., 1999), hCLC-2 (Britton et al., 2005), and a putative channel ngCLC (Fortea Verdejo, 2021); transporter homologs include hCLC-3 (Guzman et al., 2013), hCLC-4, hCLC-5 (Picollo & Pusch,…”

“The others, hCLC-3, hCLC-4, hCLC-5, hCLC-6, and hCLC-7 have been demonstrated to act as coupled chloride/proton antiporters (Guzman et al., 2013; Leisle et al., 2011; Neagoe et al., 2010; Picollo & Pusch, 2005; Scheel et al., 2005).”

“Indeed, structural changes in the dimerization interface were observed in bCLC-K (Park et al., 2017), along with positioning changes of α H in hCLC-1 relative to transporter structures (Park & MacKinnon, 2018).”

“…(Saviane et al., 1999), hCLC-2 (Britton et al., 2005), and a putative channel ngCLC (Fortea Verdejo, 2021); transporter homologs include hCLC-3 (Guzman et al., 2013), hCLC-4, hCLC-5 (Picollo & Pusch, 2005; Scheel et al., 2005), hCLC-6 (Neagoe et al., 2010), hCLC-7 (Leisle et al., 2011),…”

“Introducing a valine substitution at this position is also not sufficient to confer channel activity in CLC-ec1 (Lim & Miller, 2009), suggesting that other, global changes must be involved beyond this site.”

“For example, AF2 structure predictions were used to expand on the classification of solute carrier membrane transporter folds (Ferrada & Superti-Furga, 2022).”

“The dimerization interface is formed by helices α H, α I, α P, and α Q (Figure 1c), providing a thinned and curved hydrophobic surface that drives subunit association due to the free energy gained by burying this perturbative interface away from the lipid bilayer (Chadda et al., 2021).”

“Due to the nature of CLC-ec1 and other prokaryotic homologs lacking an aqueous pathway across the membrane, most of the conformations for transporters in the training set are expected to correspond to some type of occluded state (Khantwal et al., 2016).”

“These proteins play vital physiological roles governing the excitability of muscle and neurons (Bennetts et al., 2005; Guzman et al., 2014), acidification of lysosomes and bones (Kornak et al., 2001; Zifarelli, 2022), and acid resistance in certain bacteria (Gut et al., 2006).”

“Prior applications of DMs in the CLC family have primarily focused on comparing alternative conformations of the same sequence (Chavan et al., 2020), but their utility extends further.”

“These proteins play vital physiological roles governing the excitability of muscle and neurons (Bennetts et al., 2005; Guzman et al., 2014), acidification of lysosomes and bones (Kornak et al., 2001; Zifarelli, 2022), and acid resistance in certain bacteria (Gut et al., 2006).”

“These DDMs, report positive and negative distance deviations corresponding to regions of expansion or compression, and have been used previously to analyze defined conformational changes (Licht et al., 2024).”

“To identify how our classifier makes its predictions, we employed SHAP (SHapley Additive exPlanations) values, a method that quantifies the expected marginal contribution of each input feature to the classifier ’ s output for a single prediction (Lundberg & Lee, 2017; Winter, 2002).”

“Use of structural prediction as a tool in phylogenetic analysis is growing more common as the technology becomes more widespread and accessible (Hunker & Bleichert, 2026; Kim et al., 2025; Mishra et al., 2025).”