Mega-scale experimental analysis of protein folding stability in biology and design.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

Binding motif for RIC-3 chaperon protein in serotonin type 3A receptors.

Serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors belong to the family of pentameric ligand-gated ion channels (pLGICs) that are therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities of pLGICs' extracellular and transmembrane domains, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A subunit intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) protein. Previously, we have shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In the present study, synthetic L1-MX-based peptides and Ala-scanning identify positions W347, R349, and L353 as critical for binding to RIC-3. Complementary studies using full-length 5-HT3A subunits confirm that the identified Ala substitutions reduce the RIC-3-mediated modulation of functional surface expression. Additionally, we find and characterize a duplication of the binding motif, DWLR…VLDR, present in both the MX-helix and the transition between the ICD MA-helix and transmembrane segment M4. Analogous Ala substitutions at W447, R449, and L454 disrupt MAM4-peptide RIC-3 interactions and reduce modulation of functional surface expression. In summary, we identify the binding motif for RIC-3 in 5-HT3A subunits at two locations in the ICD, one in the MX-helix and one at the MAM4-helix transition.

High-accuracy protein model quality assessment using attention graph neural networks.

Great improvement has been brought to protein tertiary structure prediction through deep learning. It is important but very challenging to accurately rank and score decoy structures predicted by different models. CASP14 results show that existing quality assessment (QA) approaches lag behind the development of protein structure prediction methods, where almost all existing QA models degrade in accuracy when the target is a decoy of high quality. How to give an accurate assessment to high-accuracy decoys is particularly useful with the available of accurate structure prediction methods. Here we propose a fast and effective single-model QA method, QATEN, which can evaluate decoys only by their topological characteristics and atomic types. Our model uses graph neural networks and attention mechanisms to evaluate global and amino acid level scores, and uses specific loss functions to constrain the network to focus more on high-precision decoys and protein domains. On the CASP14 evaluation decoys, QATEN performs better than other QA models under all correlation coefficients when targeting average LDDT. QATEN shows promising performance when considering only high-accuracy decoys. Compared to the embedded evaluation modules of predicted ${C}_{\alpha^{-}} RMSD$ (pRMSD) in RosettaFold and predicted LDDT (pLDDT) in AlphaFold2, QATEN is complementary and capable of achieving better evaluation on some decoy structures generated by AlphaFold2 and RosettaFold. These results suggest that the new QATEN approach can be used as a reliable independent assessment algorithm for high-accuracy protein structure decoys.

Allosteric autoregulation of DNA binding via a DNA-mimicking protein domain: a biophysical study of ZNF410-DNA interaction using small angle X-ray scattering.

ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA binding affinity, compared to that of the isolated DNA binding ZF array, both in vitro and in cells. AlphaFold predicts a partially-folded N-terminal subdomain that includes a 30-residue long helix, preceded by a hairpin loop rich in acidic (aspartate/glutamate) and serine/threonine residues. This hairpin loop is predicted by AlphaFold to lie against the DNA binding interface of the ZF array. In solution, ZNF410 is a monomer and binds to DNA with 1:1 stoichiometry. Surprisingly, the single best-fit model for the experimental small angle X-ray scattering profile, in the absence of DNA, is the original AlphaFold model with the N-terminal long-helix and the hairpin loop occupying the ZF DNA binding surface. For DNA binding, the hairpin loop presumably must be displaced. After combining biophysical, biochemical, bioinformatic and artificial intelligence-based AlphaFold analyses, we suggest that the hairpin loop mimics the structure and electrostatics of DNA, and provides an additional mechanism, supplementary to sequence specificity, of regulating ZNF410 DNA binding.

Human TMPRSS2 non-catalytic ectodomain and SARS-CoV-2 S2' subunit interaction mediated SARS-CoV-2 endocytosis: a model proposal with virtual screening for potential drug molecules to inhibit this interaction.

This study proposes a novel model for integration of SARS-CoV-2 into host cell via endocytosis as a possible alternative to the prevailing direct fusion model. It is known that the SARS-CoV-2 spike protein undergoes proteolytic cleavage at S1-S2 cleavage site and the cleaved S2 domain is primed by the activated serine protease domain (SPD) of humanTMPRSS2 to become S2'. The activated SPD of TMPRSS2 is formed after it is cleaved by autocatalysis from the membrane bound non-catalytic ectodomain (hNECD) comprising of LDLRA CLASS-I repeat and a SRCR domain. It is known that the SRCR domains as well as LDLRA repeat harboring proteins mediate endocytosis of viruses and certain ligands. Based on this, we put forward a hypothesis that the exposed hNECD binds to the S2' as both are at an interaction proximity soon after S2 is processed by the SPD and this interaction may lead to the endocytosis of virus. Based on this hypothesis we have modelled the hNECD structure, followed by docking studies with the known 3D structure of S2'. The interaction interface of hNECD with S2' was further used for virtual screening of FDA-approved drug molecules and Indian medicinal plant-based compounds. We also mapped the known mutations of concern and mutations of interest on interaction interface of S2' and found that none of the known mutations map onto the interaction interface. This indicates that targeting the interaction between the hNECD of TMPRSS2 and S2' may serve as an attractive therapeutic target.Communicated by Ramaswamy H. Sarma.

Predicting the Assembly of the Transmembrane Domains of Viral Channel Forming Proteins and Peptide Drug Screening Using a Docking Approach.

A de novo assembly algorithm is provided to propose the assembly of bitopic transmembrane domains (TMDs) of membrane proteins. The algorithm is probed using, in particular, viral channel forming proteins (VCPs) such as M2 of influenza A virus, E protein of severe acute respiratory syndrome corona virus (SARS-CoV), 6K of Chikungunya virus (CHIKV), SH of human respiratory syncytial virus (hRSV), and Vpu of human immunodeficiency virus type 2 (HIV-2). The generation of the structures is based on screening a 7-dimensional space. Assembly of the TMDs can be achieved either by simultaneously docking the individual TMDs or via a sequential docking. Scoring based on estimated binding energies (EBEs) of the oligomeric structures is obtained by the tilt to decipher the handedness of the bundles. The bundles match especially well for all-atom models of M2 referring to an experimentally reported tetrameric bundle. Docking of helical poly-peptides to experimental structures of M2 and E protein identifies improving EBEs for positively charged (K,R,H) and aromatic amino acids (F,Y,W). Data are improved when using polypeptides for which the coordinates of the amino acids are adapted to the Cα coordinates of the respective experimentally derived structures of the TMDs of the target proteins.

Apigenin analogues as SARS-CoV-2 main protease inhibitors: In-silico screening approach.

The COVID-19 new variants spread rapidly all over the world, and until now scientists strive to find virus-specific antivirals for its treatment. The main protease of SARS-CoV-2 (Mpro) exhibits high structural and sequence homology to main protease of SARS-CoV (93.23% sequence identity), and their sequence alignment indicated 12 mutated/variant residues. The sequence alignment of SARS-CoV-2 main protease led to identification of only one mutated/variant residue with no significant role in its enzymatic process. Therefore, Mpro was considered as a high-profile drug target in anti-SARS-CoV-2 drug discovery. Apigenin analogues to COVID-19 main protease binding were evaluated. The detailed interactions between the analogues of Apigenin and SARS-CoV-2 Mpro inhibitors were determined as hydrogen bonds, electronic bonds and hydrophobic interactions. The binding energies obtained from the molecular docking of Mpro with Boceprevir, Apigenin, Apigenin 7-glucoside-4'-p-coumarate, Apigenin 7-glucoside-4'-trans-caffeate and Apigenin 7-O-beta-d-glucoside (Cosmosiin) were found to be -6.6, -7.2, -8.8, -8.7 and -8.0 kcal/mol, respectively. Pharmacokinetic parameters and toxicological characteristics obtained by computational techniques and Virtual ADME studies of the Apigenin analogues confirmed that the Apigenin 7-glucoside-4'-p-coumarate is the best candidate for SARS-CoV-2 Mpro inhibition.

The CBM48 domain-containing protein FLO6 regulates starch synthesis by interacting with SSIVb and GBSS in rice.

KEY MESSAGE: FLO6 is involved in starch synthesis by interacting with SSIVb and GBSS in rice. Starch synthesized and stored in plastids including chloroplasts and amyloplasts plays a vital role in plant growth and provides the major energy for human diet. However, the molecular mechanisms by which regulate starch synthesis remain largely unknown. In this study, we identified and characterized a rice floury endosperm mutant M39, which exhibited defective starch granule formation in pericarp and endosperm, accompanied by the decreased starch content and amylose content. The abnormal starch accumulation in M39 pollen grains caused a significant decrease in plant fertility. Chloroplasts in M39 leaves contained no or only one large starch granule. Positional cloning combined with complementary experiment demonstrated that the mutant phenotypes were restored by the FLOURY ENDOSPERM6 (FLO6). FLO6 was generally expressed in various tissues, including leaf, anther and developing endosperm. FLO6 is a chloroplast and amyloplast-localized protein that is able to bind to starch by its carbohydrate-binding module 48 (CBM48) domain. Interestingly, we found that FLO6 interacted with starch synthase IVb (SSIVb) and granule-bound starch synthase (GBSSI and GBSSII). Together, our results suggested that FLO6 plays a critical role in starch synthesis through cooperating with several starch synthesis enzymes throughout plant growth and development.

The duality of PRDM proteins: epigenetic and structural perspectives.

PRDF1 and RIZ1 homology domain containing (PRDMs) are a subfamily of Krüppel-like zinc finger proteins controlling key processes in metazoan development and in cancer. PRDMs exhibit unique dualities: (a) PR domain/ZNF arrays-their structure combines a SET-like domain known as a PR domain, typically found in methyltransferases, with a variable array of C2H2 zinc fingers (ZNF) characteristic of DNA-binding transcription factors; (b) transcriptional activators/repressors-their physiological function is context- and cell-dependent; mechanistically, some PRDMs have a PKMT activity and directly catalyze histone lysine methylation, while others are rather pseudomethyltransferases and act by recruiting transcriptional cofactors; (c) oncogenes/tumor suppressors-their pathological function depends on the specific PRDM isoform expressed during tumorigenesis. This duality is well known as the 'Yin and Yang' of PRDMs and involves a complex regulation of alternative splicing or alternative promoter usage, to generate full-length or PR-deficient isoforms with opposing functions in cancer. In conclusion, once their dualities are fully appreciated, PRDMs represent a promising class of targets in oncology by virtue of their widespread upregulation across multiple tumor types and their somatic dispensability, conferring a broad therapeutic window and limited toxic side effects. The recent discovery of a first-in-class compound able to inhibit PRDM9 activity has paved the way for the identification of further small molecular inhibitors able to counteract PRDM oncogenic activity.

The inhibitory effects of PGG and EGCG against the SARS-CoV-2 3C-like protease.

The coronavirus disease (COVID-19) pandemic, resulting from human-to-human transmission of a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has led to a global health crisis. Given that the 3 chymotrypsin-like protease (3CLpro) of SARS-CoV-2 plays an indispensable role in viral polyprotein processing, its successful inhibition halts viral replication and thus constrains virus spread. Therefore, developing an effective SARS-CoV-2 3CLpro inhibitor to treat COVID-19 is imperative. A fluorescence resonance energy transfer (FRET)-based method was used to assess the proteolytic activity of SARS-CoV-2 3CLpro using intramolecularly quenched fluorogenic peptide substrates corresponding to the cleavage sequence of SARS-CoV-2 3CLpro. Molecular modeling with GEMDOCK was used to simulate the molecular interactions between drugs and the binding pocket of SARS-CoV-2 3CLpro. This study revealed that the Vmax of SARS-CoV-2 3CLpro was about 2-fold higher than that of SARS-CoV 3CLpro. Interestingly, the proteolytic activity of SARS-CoV-2 3CLpro is slightly more efficient than that of SARS-CoV 3CLpro. Meanwhile, natural compounds PGG and EGCG showed remarkable inhibitory activity against SARS-CoV-2 3CLpro than against SARS-CoV 3CLpro. In molecular docking, PGG and EGCG strongly interacted with the substrate binding pocket of SARS-CoV-2 3CLpro, forming hydrogen bonds with multiple residues, including the catalytic residues C145 and H41. The activities of PGG and EGCG against SARS-CoV-2 3CLpro demonstrate their inhibition of viral protease activity and highlight their therapeutic potentials for treating SARS-CoV-2 infection.

Probing Protein-protein Interactions and Druggable Site Identification: Mechanistic Binding Events Between Ubiquitin and Zinc Finger with UFM1-specific Peptidase Domain Protein (ZUFSP).

BACKGROUND: Deubiquitinating enzymes (DUBs) protein family have been implicated in some deregulated pathways involved in carcinogeneses, such as cell cycle, gene expression, and DNA damage response (DDR). Zinc finger with UFM1-specific peptidase domain protein (ZUFSP) is one of the recently discovered members of the DUBs. OBJECTIVES: To identify and cross-validate the ZUFSP binding site using the bioinformatic tools, including SiteMap&Metapocket, respectively. To understand the molecular basis of complementary ZUFSP-Ub interaction and associated structural events using MD Simulation. METHODS: In this study, four binding pockets were predicted, characterized, and cross-validated based on physiochemical features such as site score, druggability score, site volume, and site size. Also, a molecular dynamics simulation technique was employed to determine the impact of ubiquitin-binding on ZUFSP. RESULTS: Site 1 with a site score 1.065, Size 102, D scores 1.00, and size volume 261 was predicted to be the most druggable site. Structural studies revealed that upon ubiquitin-binding, the motional movement of ZUFSP was reduced when compared to the unbound ZUFSP. Also, the ZUFSP helical arm (ZHA) domain orient in such a way that it moves closer to the Ub; this orientation enables the formation of a UBD which is very peculiar to ZUFSP. CONCLUSION: The impact of ubiquitin on ZUFSP movement and the characterization of its predicted druggable site can be targeted in the development of therapeutics.

Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease.

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

Metal-Specificity Divergence between Metallothioneins of Nerita peloronta (Neritimorpha, Gastropoda) Sets the Starting Point for a Novel Chemical MT Classification Proposal.

Metallothioneins' (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal-MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.

Protein domain-based prediction of drug/compound-target interactions and experimental validation on LIM kinases.

Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

Receptor-binding domain of SARS-CoV-2 spike protein efficiently inhibits SARS-CoV-2 infection and attachment to mouse lung.

COVID-19, caused by a novel coronavirus, SARS-CoV-2, poses a serious global threat. It was first reported in 2019 in China and has now dramatically spread across the world. It is crucial to develop therapeutics to mitigate severe disease and viral spread. The receptor-binding domains (RBDs) in the spike protein of SARS-CoV and MERS-CoV have shown anti-viral activity in previous reports suggesting that this domain has high potential for development as therapeutics. To evaluate the potential antiviral activity of recombinant SARS-CoV-2 RBD proteins, we determined the RBD residues of SARS-CoV-2 using a homology search with RBD of SARS-CoV. For efficient expression and purification, the signal peptide of spike protein was identified and used to generate constructs expressing recombinant RBD proteins. Highly purified RBD protein fused with the Fc domain of human IgG showed potent anti-viral efficacy, which was better than that of a protein fused with a histidine tag. Intranasally pre-administrated RBD protein also inhibited the attachment of SARS-COV-2 to mouse lungs. These findings indicate that RBD protein could be used for the prevention and treatment of SARS-CoV-2 infection.

Hyperthermostability of prawn ferritin nanocage facilitates its application as a robust nanovehicle for nutraceuticals.

The favorable physicochemical properties are essential for the application of protein-based nanovehicles in the field of biomaterials. Herein, we found that the thermal stability of Marsupenaeus japonicus ferritin (MjFer) (Tm = 109.1 ± 0.4 °C) is markedly higher than human H-chain ferritin (HuHF) (Tm = 87.7 ± 0.3 °C), although they share a high structural similarity. Multiple results indicated that the promoted thermal stability of MjFer is mainly derived from the salt bridges located at the C3 interface. Consequently, MjFer exhibits strong protective effects on encapsulated curcumin upon exposure at high temperatures. In contrast, most of the curcumin loaded HuHF composites precipitated rapidly under the same conditions. These findings elucidated the molecular mechanism of the hyperthermostability of MjFer and illustrated that MjFer could act as a robust insulation nanocarrier for bioactive compounds against various thermal treatments.

Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins.

Motifs within proteins help us categorize their functions. Intrinsically disordered proteins (IDPs) are rich in short linear motifs, conferring them many different roles. IDPs are also frequently highly charged and, therefore, likely to interact with ions. Canonical calcium-binding motifs, such as the EF-hand, often rely on the formation of stabilizing flanking helices, which are a key characteristic of folded proteins, but are absent in IDPs. In this study, we probe the existence of a calcium-binding motif relevant to IDPs. Upon screening several carefully selected IDPs using NMR spectroscopy supplemented with affinity quantification by colorimetric assays, we found calcium-binding motifs in IDPs which could be categorized into at least two groups-an Excalibur-like motif, sequentially similar to the EF-hand loop, and a condensed-charge motif carrying repetitive negative charges. The motifs show an affinity for calcium typically in the ~100 µM range relevant to regulatory functions and, while calcium binding to the condensed-charge motif had little effect on the overall compaction of the IDP chain, calcium binding to Excalibur-like motifs resulted in changes in compaction. Thus, calcium binding to IDPs may serve various structural and functional roles that have previously been underreported.

Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes.

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

Structural basis for Ca2+ activation of the heteromeric PKD1L3/PKD2L1 channel.

The heteromeric complex between PKD1L3, a member of the polycystic kidney disease (PKD) protein family, and PKD2L1, also known as TRPP2 or TRPP3, has been a prototype for mechanistic characterization of heterotetrametric TRP-like channels. Here we show that a truncated PKD1L3/PKD2L1 complex with the C-terminal TRP-fold fragment of PKD1L3 retains both Ca2+ and acid-induced channel activities. Cryo-EM structures of this core heterocomplex with or without supplemented Ca2+ were determined at resolutions of 3.1 Å and 3.4 Å, respectively. The heterotetramer, with a pseudo-symmetric TRP architecture of 1:3 stoichiometry, has an asymmetric selectivity filter (SF) guarded by Lys2069 from PKD1L3 and Asp523 from the three PKD2L1 subunits. Ca2+-entrance to the SF vestibule is accompanied by a swing motion of Lys2069 on PKD1L3. The S6 of PKD1L3 is pushed inward by the S4-S5 linker of the nearby PKD2L1 (PKD2L1-III), resulting in an elongated intracellular gate which seals the pore domain. Comparison of the apo and Ca2+-loaded complexes unveils an unprecedented Ca2+ binding site in the extracellular cleft of the voltage-sensing domain (VSD) of PKD2L1-III, but not the other three VSDs. Structure-guided mutagenic studies support this unconventional site to be responsible for Ca2+-induced channel activation through an allosteric mechanism.