Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin ␣ 2 ␤ 1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly 3 Xaa (Xaa ‫؍‬ Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin ␣ 2 ␤ 1binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin ␣ 2 ␤ 1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin ␣ 2-inserted (␣ 2 I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly 3 Xaa substitutions differentially disrupt the CMP-␣ 2 I interaction. The Gly 3 Arg substitution destabilized CMP-␣ 2 I side-chain interactions, and the Gly 3 Val change broke the essential Mg 2؉ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa, as evidenced by its loss or downregulation in high-grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial-mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. Cell Commun Signal. 2019). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggests the possibility that it plays a role in EMT by directly regulating transcriptional activity by DNA binding. To examine this hypothesis, we set out to analyze ABI1-DNA binding. Structural NMR studies and in-vitro protein-DNA binding assays confirmed ABI1 binding to DNA regulated by alternative spliced ABI1 Exon 4 region located on the C-terminus of ABI1 HHR. RNA-sequencing data and PCa cell lines qPCR assays reveal the alternative ABI1 Exon 4 spliced-in is enriched in high Gleason-scored PCa samples. ABI1 and STAT3 co-regulate their nuclear vs. cytoplasm localization, and the subsequent functional studies using PCa lines expressing wild type or HHR Exon 4 deletion mutant of ABI1 demonstrated that HHR regulates the STAT3 DNA binding patterns, STAT3 mediated chromatin structure programming as well as its transcription activities. We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression. Citation Format: Xiang Li, Neeru Arya, Baylee A. Porter, Allysa P. Kemraj, Xuesen Dong, Dominique Frueh, Alaji Bah, Leszek Kotula. ABI1 regulates STAT3 transcription through a DNA binding activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3725.

Background: Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa as evidenced by its loss or downregulation in high grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial-mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. 2019 Cell Commun Signal). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggested the possibility that it plays a role in EMT through directly regulating transcriptional activity by DNA binding. Methods: To examine this hypothesis we set out to analyze ABI1-DNA binding. We purified ABI1 HHR and demonstrated its in vitro interactions with different homeobox DNA-binding consensus of double-stranded DNA sequences. We created DU145 ABI1 CRISPR KO cell line and rescue ABI1 expression using retroviral transfection with either ABI1 wild type or Abi1 HHR mutants. DU145 cell lines STAT3 cellular localization was studied by Immunofluorescence staining and the STAT3 transcription activities was studied by Firefly Luciferase Assays with STAT3 specific luciferase reporter plasmid. Results: We found that the presence of alternatively spliced Exon 4-encoded sequence, located in the C-terminus of HHR, regulates binding of ABI1 to DNA. Structural NMR studies confirmed ABI1 binding to DNA. Subsequent functional studies using DU145 CRISPR KO cell lines expressing wild type or HHR mutants of ABI1 demonstrated that HHR regulates the nuclear localization and transcriptional activity of STAT3. Conclusion: We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression by the ABI1 HHR mediated DNA binding mechanism. Funding resources: NCI R01 CA161018; R21 CA260381 Conflicts of Interest Disclosure Statement: The authors declare no conflicts of interest. Citation Format: Xiang Li, Baylee Porter, Allysa Kemraj, Alaji Bah, Marcin Kortylewski, Leszek Kotula. ABI1 regulates transcriptional activity of STAT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2364.

Hyperpolarization-activated channels, cyclic nucleotide-gated (HCN) channels show an inverted voltage response compared to virtually all other voltage-gated ion channels (VGICs), opening on hyperpolarization rather than depolarization. The basic motions of voltage sensing and pore gating are thought to be conserved, implying that these domains are inversely coupled in HCN channels. Although the structure of the HCN1 channel was recently solved, the structural element(s) responsible for the inverted gating polarity of HCN are not known. We used a structure-guided protein engineering approach to systematically assembled an array of mosaic channels between HCN1 and the depolarization-activated EAG that display the full complement of voltageactivation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage-sensing domain of the HCN channel has an intrinsic ability to drive pore opening in either direction. Specific contacts at the voltage sensor-pore interface and unique interactions near the pore gate force the HCN channel into a hERG-like inactivated state, thereby obscuring their opening upon depolarization. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity. 85-PlatMolecular Simulations of Ion Permeation, Gating and Selectivity in K D Potassium channels play a pivotal role in many biological functions, such as formation of the membrane potential and mediating electrical signals in excitable cells (e.g. neurons). Structural and functional studies revealed main features of these channels, including rapid and selective K þ ion permeation through a narrow selectivity filter (SF), channel gating at the helix bundle crossing (activation gate), and distinct gating at the SF, termed C-type inactivation. Despite such insights, the molecular mechanisms of permeation and gating phenomena remain largely unknown, and are further complicated by differences exhibited in numerous, structurally distinct members of the potassium channel family. Nowadays, Molecular Dynamics (MD) simulations allow studying thousands of individual ion permeation events on the atomistic scale, that directly correspond to experimentally measured macroscopic currents. Here, we will show simulations of several potassium channels (KcsA, MthK, NaK2K, Kv1.2 and TRAAK), all sharing nearly identical SFs. Our simulations show that all studied K þ channels achieve their high conductance rates by permeation of 'naked' K þ ions through the SF. Simultaneously, the interactions of fully desolvated potassium ions in the SF efficiently exclude any sodium ions from permeation, due to their higher dehydration penalty. Moreover, we will present simulation data that link channel and SF conformations with the ion permeation rates, thus explaining channel gating at the molecular level. 86-PlatVoltage-Sensing Residues in the Voltage Sensor of the BK Channel Voltage-and Ca 2þ -activated K þ (BK) channels are modular proteins with allosteric ...