Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

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Biophysical Society Thematic Meetings

PROGRAM & ABSTRACTS

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy Thessaloniki, Greece | November 9–13, 2025

Organizing Committee

Matthias Buck, Case Western Reserve University, USA Zoe Cournia, Biomedical Research Foundation Academy of Athens, Greece Alemayehu Gorfe, University of Texas Health Science Center at Houston, USA Themis Lazaridis, The City College of New York, USA

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Welcome Letter

November 2025

Dear Colleagues,

We would like to welcome you to the Biophysical Society Thematic Meeting entitled, Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy . Membrane proteins are integral to signal transduction and many other critical cellular processes. Peripheral membrane proteins (PMPs) are a class of membrane proteins that attach to the lipid bilayer acting on the lipid-water interface in contrast to transmembrane proteins, which are fully embedded in the cell membrane. This meeting will focus on recent advances on PMP studies, including their interaction with membranes and roles in health and disease, as well as their targeting for drug design. The presentations will include both experimental and computational work, with emphasis on conceptual and technological limitations to a better structural and functional understanding of PMPs, and how to overcome those limitations in future. Overall, this conference will feature 23 posters, 39 lectures, and bring together over 50 scientists from a wide range of backgrounds and expertise. We hope that this meeting will not only provide a place to share your recent findings, but also to help promote new collaborations, helpful discussions, and future connections. We invite you all to actively take part in the discussions following each talk, the poster sessions, and the informal exchanges that will be possible during the coffee breaks and meals. We also hope that you will also enjoy the beautiful city of Thessaloniki!

The Organizing Committee Matthias Buck Zoe Cournia

Alemayehu Gorfe Themis Lazaridis

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Meeting Code of Conduct

Biophysical Society Code of Conduct, Anti-Harassment Policy The Biophysical Society (BPS) is committed to providing an environment that encourages the free expression and exchange of scientific ideas. As a global, professional Society, the BPS is committed to the philosophy of equal opportunity and respectful treatment for all, regardless of national or ethnic origin, religion or religious belief, gender, gender identity or expression, race, color, age, marital status, sexual orientation, disabilities, veteran status, or any other reason not related to scientific merit. All BPS meetings and BPS-sponsored activities promote an environment that is free of inappropriate behavior and harassment by or toward all attendees and participants of Society events, including speakers, organizers, students, guests, media, exhibitors, staff, vendors, and other suppliers. BPS expects anyone associated with an official BPS-sponsored event to respect the rules and policies of the Society, the venue, the hotels, and the city. Definition of Harassment The term “harassment” includes but is not limited to epithets, unwelcome slurs, jokes, or verbal, graphic or physical conduct relating to an individual’s race, color, religious creed, sex, national origin, ancestry, citizenship status, age, gender or sexual orientation that denigrate or show hostility or aversion toward an individual or group. Sexual harassment refers to unwelcome sexual advances, requests for sexual favors, and other verbal or physical conduct of a sexual nature. Behavior and language that are welcome/acceptable to one person may be unwelcome/offensive to another. Consequently, individuals must use discretion to ensure that their words and actions communicate respect for others. This is especially important for those in positions of authority since individuals with lower rank or status may be reluctant to express their objections or discomfort regarding unwelcome behavior. It does not refer to occasional compliments of a socially acceptable nature. It refers to behavior that is not welcome, is personally offensive, debilitates morale, and therefore, interferes with work effectiveness. The following are examples of behavior that, when unwelcome, may constitute sexual harassment: sexual flirtations, advances, or propositions; verbal comments or physical actions of a sexual nature; sexually degrading words used to describe an individual; a display of sexually suggestive objects or pictures; sexually explicit jokes; unnecessary touching. Attendees or participants who are asked to stop engaging in harassing behavior are expected to comply immediately. Anyone who feels harassed is encouraged to immediately inform the alleged harasser that the behavior is unwelcome. In many instances, the person is unaware that their conduct is offensive and when so advised can easily and willingly correct the conduct so that it does not reoccur. Anyone who feels harassed is NOT REQUIRED to address the person believed guilty of inappropriate treatment. If the informal discussion with the alleged harasser is unsuccessful in remedying the problem or if the complainant does not feel comfortable with such an approach, they can report the behavior as detailed below. Reported or suspected occurrences of harassment will be promptly and thoroughly investigated. Following an investigation, BPS will immediately take any necessary and appropriate action. BPS will not permit or condone any acts of retaliation against anyone who files harassment complaints or cooperates in the investigation of same. Reporting a Violation Violations of this Conduct Policy should be reported immediately. If you feel physically unsafe or believe a crime has been committed, you should report it to the police immediately. To report a violation to BPS:

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Meeting Code of Conduct

 You may do so in person to BPS senior staff at Thematic Meetings, BPS Conferences, or other BPS events.

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Reported or suspected occurrences of harassment will be promptly and thoroughly investigated per the procedure detailed below. Following an investigation, BPS will immediately take any necessary and appropriate action. BPS will not permit or condone any acts of retaliation against anyone who files harassment complaints or cooperates in the investigation of same. Investigative Procedure All reports of harassment or sexual harassment will be treated seriously. However, absolute confidentiality cannot be promised nor can it be assured. BPS will conduct an investigation of any complaint of harassment or sexual harassment, which may require limited disclosure of pertinent information to certain parties, including the alleged harasser. Once a complaint of harassment or sexual harassment is received, BPS will begin a prompt and thorough investigation. Please note, if a complaint is filed anonymously, BPS may be severely limited in our ability to follow-up on the allegation.  An impartial investigative committee, consisting of the current President, President-Elect, and Executive Officer will be established. If any of these individuals were to be named in an allegation, they would be excluded from the committee.  The committee will interview the complainant and review the written complaint. If no written complaint exists, one will be requested.  The committee will speak to the alleged offender and present the complaint.  The alleged offender will be given the opportunity to address the complaint, with sufficient time to respond to the evidence and bring his/her own evidence.  If the facts are in dispute, the investigative team may need to interview anyone named as witnesses.  The investigative committee may seek BPS Counsel’s advice.  Once the investigation is complete, the committee will report their findings and make recommendations to the Society Officers.  If the severity of the allegation is high, is a possible repeat offense, or is determined to be beyond BPS’s capacity to assess claims and views on either side, BPS may refer the case to the alleged offender’s home institution (Office of Research Integrity of similar), employer, licensing board, or law enforcement for their investigation and decision. Disciplinary Actions Individuals engaging in behavior prohibited by this policy as well as those making allegations of harassment in bad faith will be subject to disciplinary action. Such actions range from a written warning to ejection from the meeting or activity in question without refund of registration fees, being banned from participating in future Society meetings or Society-sponsored activities, being expelled from membership in the Society, and reporting the behavior to their employer or calling the authorities. In the event that the individual is dissatisfied with the results of the investigation, they may appeal to the President of the Society. Any questions regarding this policy should be directed to the BPS Executive Officer or other Society Officer.

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Table of Contents

Table of Contents

General Information……………………………………………………………………………....1 Program Schedule..……………………………………………………………………………….3 Speaker Abstracts………………………………………………………………………………...8 Poster Sessions…………………………………………………………………………………...39

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

General Information

GENERAL INFORMATION

Registration/Information Location and Hours The meeting will take place at the Aristotle University Research Dissemination Center (KEDEA), located at 3 rd Septemvriou Str., Thessaloniki 456 36, Greece. To pick up your badge and meeting materials, please visit the BPS Registration Desk, located in the Conference Hall III Foyer during the following times: Sunday, November 9 16:00 – 18:00 Monday, November 10 8:30 – 18:00 Tuesday, November 11 8:30 – 18:00 Wednesday, November 12 13:00 – 18:00 Thursday, November 13 8:30 – 15:00 Instructions for Presentations (1) Presentation Facilities: A data projector will be available in Conference Hall III, located on Level -1. Speakers are required to bring their own laptops and adaptors. It is recommended to have a backup of the presentation on a USB drive in case of any unforeseen circumstances. Speakers are advised to preview their final presentations before the start of each session. (2) Poster Session: 1) All poster sessions will be held in the Foyer (-1). 2) A display board measuring 91 cm by 182 cm (portrait orientation) will be provided for each poster. Poster boards are numbered according to the same numbering scheme as listed in the E-book. 3) Posters may only be mounted using masking tape or painters tape. BPS will provide tape for poster mounting. 4) There will be formal poster presentations on Monday and Tuesday from 14:30 – 16:00. Please refer to the daily schedule for your formal presentation date and time. Ninety (90) minutes have been allotted for poster presentations each day. Presenting authors with odd-numbered poster boards should present during the first 45 minutes, and those with even-numbered poster boards should present during the last 45 minutes. 5) During the assigned poster presentation sessions, presenters are requested to remain in front of their poster boards to meet with attendees. 6) All posters left uncollected at the end of the meeting will be discarded.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

General Information

Note Pads/Pens Society pens will be provided, however please bring your own note pad. Meals, Coffee Breaks, and Socials

The opening mixer, coffee breaks and luncheons will be served in the Foyer (-1). The Wednesday evening banquet dinner will be held at 18:30 (6:30 PM) in the Makedonia Room of the Electra Palace Hotel, located at 9 Aristotelous Sq., 546 24 Thessaloniki. Smoking Please be advised that the Aristotle University Research Dissemination Center (KEDEA) is a non Name badges will be given to you when you check-in at the Registration Desk in the Conference Hall III Foyer. Badges are required to enter all scientific sessions, poster sessions, and social functions. Please wear your badge throughout the conference. Internet Wi-Fi will be provided at the venue. Information will be available at the registration desk. On-Site Contact Information If you have any further requirements during the meeting, please contact the meeting staff at the registration desk from November 9-13 during registration hours. In case of emergency, you may contact the following: Dorothy Chaconas Phone: 301-785-0802 Email: dchaconas@biophysics.org Erica Bellavia Phone: 571-435-7669 Email: ebellavia@biophysics.org smoking facility. Name Badges

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Daily Schedule

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy Thessaloniki, Greece November 9-13, 2025 All scientific sessions will be held in Conference Hall III unless otherwise noted.

Sunday, November 9, 2025

16:00 – 18:00

Registration/Information

Conference Hall III Foyer

18:00 – 19:30

Welcome Reception

Foyer (-1)

Monday, November 10, 2025 8:30 – 18:00

Registration/Information

Conference Hall III Foyer

8:50 – 9:00

Matthias Buck, Case Western Reserve University, USA & Zoe Cournia, Biomedical Research Foundation Academy of Athens, Greece Welcome & Opening Remarks Membrane Interaction of Kinases Chair: Alemayehu Gorfe, University of Texas Health Science Center at Houston, USA Kalina Hristova, Johns Hopkins University, USA Biophysics of Growth Factor Receptor Organization and Signaling in the Membrane Adam Smith, Texas Tech University, USA Anionic Lipids Inhibit the Catalytic Activity of the Membrane Proximal EGFR Kinase Domain

Session I

9:00 – 9:30

9:30 – 10:00

10:00 – 10:20

Matthias Buck, Case Western Reserve University, USA Plexin and EPH Receptors: The View

10:20 – 10:50

Coffee Break

Foyer (-1)

10:50 – 11:20

Daniel Abankwa, University of Luxembourg, Luxembourg SPRY2 Interacts with Active RAS to Modulate Its Membrane Organization Helgi Ingólfsson, Lawrence Livermore National Laboratory, USA Capturing Lipid-Protein Interactions During RAS-RAF Activation Using Machine Learning-Driven Multiscale Simulations Rachel McAllister, Yale University, USA* Novel Two-Step Peripheral Membrane Recruitment Mechanism of Bruton's Tyrosine Kinase Through Native Mass Spectrometry

11:20 – 11:50

11:50 – 12:10

12:10 – 13:10

Lunch

Foyer (-1)

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Daily Schedule

Session II

Membrane Remodeling Chair: Themis Lazaridis, The City College of New York, USA Patricia Bassereau, Institute Curie A Minimal Human ESCRT-III System to Mimic HIV-1 Detachment Winfried Weissenhorn, Institute of Structural Biology, France Constricting and Cleaving Membrane Necks with ESCRT-III and VPS4

13:10 – 13:40

13:40 – 14:10

14:10 – 14:30

Dirk Schneider, Johannes Gutenberg University, Germany* Membrane Interaction of a Prokaryotic ESCRT-III Protein

14:30 – 16:00

Coffee Break & Poster Session I

Foyer (-1)

16:00 – 16:30

Phillip Stansfeld, University of Warwick, United Kingdom Modelling Lipid Interactions at Membrane Interfaces with MEMPRO and CCD2MD Anne Kenworthy, University of Virginia, USA Controlling Cells from Deep Inside the Membrane: The Case of Caveolins Korbinian Liebl, Technical University of Munich, Germany* Membrane Remodeling by the Collective Action of Caveolin-1

16:30 – 17:00

17:00 – 17:20

17:20 – 18:00

Round Table Discussion

Tuesday, November 11, 2025 8:30 – 18:00

Registration/Information

Conference Hall III Foyer

Session III

Protein Membrane Interfaces and Membrane Lipid Composition Chair: Matthias Buck, Case Western Reserve University, USA Karen Fleming, Johns Hopkins University, USA Energetics of Side Chain Partitioning Across the Bilayer Interface Linda Columbus, University of Virginia, USA Impact of Membrane Compositions on Bacterial Membrane Protein Structure, Dynamics, and Function Kirill Grushin, Yale University, USA* Structural Insights into Munc13-1 Self-Assemblies on Lipid Bilayers from Cryo Electron Tomography

9:00 – 9:30

9:30 – 10:00

10:00 – 10:20

10:20 – 10:50

Coffee Break

Foyer (-1)

10:50 – 11:20

Nathalie Reuter, University of Bergen, Norway Membranes as Allosteric Effectors for Lipid Transfer Proteins Xiaolin Cheng, The Ohio State University, USA Integrative Modeling of Sterol Recognition by the SCAP Protein

11:20 – 11:50

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Daily Schedule

11:50 – 12:10

Alexandre Chenal, Institute Pasteur, France* Structure and Membrane Translocation Process of the Bordetella Pertussis CYAA Toxin

12:10 – 13:10

Lunch

Foyer (-1)

Session IV

Transporters, Etc. Chair: Zoe Cournia, Biomedical Research Foundation Academy of Athens, Greece Phil Biggin, University of Oxford, United Kingdom From Sequence to Mechanism: Insights Into Proton Coupling in Cystinosin from Multiscale Molecular Simulations Marco De Vivo, Italian Institute of Technology, Italy Decoding Biochemical Complexity with Simulations and AI-Enhanced Sampling Vinodhini Selvanayanaran, Indian Institute of Sciences, Bengaluru, India* Divergent Breathings Modes of EHD Paralogs: Insights From Molecular Simulations and Implications in Membrane Remodeling

13:10 – 13:40

13:40 – 14:10

14:10 – 14:30

14:30 – 16:00

Coffee Break & Poster Session II

Foyer (-1)

16:00 – 16:30

Paolo Carloni, Forschungszentrum Jülich GmbH, Germany Multiscale, ML-Assisted Simulations on Neuronal Membrane Proteins

16:30 – 17:00

Nir Ben-Tal, Tel Aviv University, Israel An AI-Based Holistic View of the Protein Universe

17:00 – 17:30

Anna Shnyrova Zhadan, University of the Basque Country, Spain Membrane Fusion Mediated by Classical Dynamin Machinery

17:30 – 18:00

Round Table Discussion

Wednesday, November 12, 2025 8:30 – 13:00

Free Time/Lunch on Own

13:00 – 18:00

Registration/Information

Conference Hall III Foyer

Session V

Inhibitors Targeting Membrane Proteins and Protein-Membrane Interfaces Chair: Matthias Buck, Case Western Reserve University, USA Ivet Bahar, Stony Brook University, USA Ferroptotic Interactions of Peripheral Membrane Proteins with Small Molecules and Lipids Zoe Cournia, Biomedical Research Foundation Academy of Athens, Greece Describing Protein Conformational Landscapes at the Protein-Membrane Interface: Implications for Drug Design Peter Chung, University of Chicago, USA* Deciphering the Organelle-Targeting Specificity of Inducible Amphipathic Helices Through a Recombinant Protein Platform

13:30 – 14:00

14:00 – 14:30

14:30 – 14:50

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Daily Schedule

14:50 – 15:20

Round Table Discussion

15:20 – 15:50

Alemayehu Gorfe, University of Texas Health Science Center at Houston, USA Characterizing the Membrane Orientation Dynamics of RHEB and Its Functional Consequences Matteo Dal Peraro, EPFL, Switzerland Unraveling the Mechanistic Diversity of Peripheral Proteins Binding to Membranes Through Integrative Approaches Diomedes Logothetis, Northeastern University, USA* Structural Understanding of G Protein GA3NG of K+ Channels in Brain and Heart

15:50 – 16:20

16:20 – 16:40

16:40 – 18:00

Free Time

18:30 – 20:30

Banquet Dinner

Electra Palace Hotel

Thursday, November 13, 2025 8:30 – 15:00

Registration/Information

Conference Hall III Foyer

Session VI

Endo-Membrane Proteins Chair: Alemayehu Gorfe, University of Texas Health Science Center at Houston, USA Stefano Vanni, University of Fribourg, Switzerland Peripheral Proteins as Master Regulators of Membrane Organization and Composition

9:00 – 9:30

9:30 – 10:00

Francisco Barrera, University of Tennessee at Knoxville, USA The Journey to the Membrane of Candidalysin, Step-By-Step

10:00 – 10:20

Mahmoud Moradi, University of Arkansas, USA* Molecular Dynamics Study of Sphingosine Kinase 1 (SK1) Regulation at the Membrane Interface

10:20 – 10:50

Coffee Break

Foyer (-1)

10:50 – 11:20

Antoine Taly, CNRS, France Prediction of A. Thaliana’s MCTP Structure, Embedding in the Endoplasmic Reticulum and Interaction with the Plasma Membrane

11:20 – 11:50

Themis Lazaridis, The City College of New York, USA Curvature Generation by the Caveolin 8S Complex

11:50 – 12:10

Dylan Suriadinata, Texas A&M University, USA* Bridging Protein TTR-53 Mediates Phosphatidylinositol Phosphate Signaling for Cell Corpse Clearance

12:10 – 12:30

Round Table Discussion

12:30 – 13:30

Lunch

Foyer (-1)

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Daily Schedule

Session VII

Receptors, Channels, and Translocators Chair: Zoe Cournia, Biomedical Research Foundation Academy of Athens, Greece Maria Kurnikova, Carnegie Mellon University, USA Conformational Ensembles and Conductivity Modeling in Tetrameric Ion Channel Receptors Syma Khalid, University of Oxford, United Kingdom Computational Microbiology: Multiscale Simulations of Membrane Active Peptides Agata Witkowska, FMP, Germany* Mechanical Control of Neurotransmission via a Disordered Domain of an Endocytic Protein Alemayehu Gorfe, University of Texas Health Science Center at Houston, USA & Themis Lazaridis, The City College of New York, USA Closing Remarks

13:30 – 14:00

14:00 – 14:30

14:30 – 14:50

14:50 – 15:00

*Contributed talks selected from among submitted abstract

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Speaker Abstracts

SPEAKER ABSTRACTS

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

BIOPHYSICS OF GROWTH FACTOR RECEPTOR SIGNALING IN THE MEMBRANE Kalina Hristova ; 1 Johns Hopkins University, Baltimore, MD, USA Microscopic GM1-enriched domains can form, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase, in response to its ligand ephrinA1-Fc. The GM1 enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantified the degree of EphA2 segregation and we studied the initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains using Forster Resonance Energy transfer and Number and Brightness. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. Given the critical role of EphA2 organization for signaling, we are using MINFLUX imaging as we seek detailed views of the EphA2-enriched and -depleted domains and mechanistic insights into oligomerization and domain formation. ANIONIC LIPIDS INHIBIT THE CATALYTIC ACTIVITY OF THE MEMBRANE PROXIMAL EGFR KINASE DOMAIN Adam W Smith 1 ; Ronald Villaber 1 ; 1 Texas Tech University, Chemistry & Biochemistry, Lubbock, TX, USA The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase whose activity is regulated not only by ligand binding and dimerization, but also by its local membrane environment. While the role of lipids in EGFR clustering and signaling has been inferred from cell-based and computational studies, direct biochemical evidence for lipid-mediated regulation of EGFR catalytic activity has been lacking. Here, we reconstitute a catalytically active intracellular domain of EGFR on synthetic liposomes, enabling us to isolate the effects of specific lipid species on kinase activity. We show that both phosphatidylinositol 4,5 bisphosphate (PIP 2 ) and phosphatidylserine (PS) act as reversible, concentration-dependent inhibitors of EGFR activity, exhibiting mixed-mode inhibition with a predominant noncompetitive component. These findings suggest that anionic phospholipids can stabilize inactive conformations of the kinase domain through electrostatic interactions, potentially contributing to autoinhibition in the absence of ligand. Our results establish a minimal, membrane-based system for dissecting lipid-protein interactions that regulate EGFR and other peripheral membrane proteins. This work has the potential to resolve the chemical details of cell signaling and to improve therapeutic targeting strategies.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

PLEXIN AND EPH RECEPTORS: THE VIEW FROM PROTEIN BIOPHYSICS ON PROTEIN-MEMBRANE INTERACTIONS Matthias Buck 1 ; Amita Sahoo 1 ; Pravesh Shrestha 1 ; Nisha Bhattarai 1 ; 1 Case Western Reserve University, Physiology and Biophysics, Cleveland, OH, USA Cell migration is guided by signals from transmembrane receptors, notably plexins and Eph receptors, which integrate protein–protein and protein–membrane interactions to control cytoskeletal dynamics. Recent biophysical and computational studies have provided structural and mechanistic insight into how these large receptors orchestrate signaling. Molecular dynamics simulations revealed that the Rho GTPase binding domain (RBD) of plexins engages Rac1 and Rnd1 with distinct isoform-specific contact dynamics, highlighting how subtle changes modulate signaling outcomes (Zhang & Buck, 2017). Downstream, the Rap1b GTPase substrate is regulated allosterically: conformational fluctuations from the transmembrane and juxtamembrane domains propagate into the intracellular GAP module (Li et al., 2021; Bhattarai et al., 2025). Coarse-grained and atomistic simulations support this allosteric view, showing how the plexin transmembrane helix samples alternative dimeric states that can tune catalytic activity (Sahoo et al., 2023). For EphA2, complementary studies demonstrate how the membrane environment itself directly regulates receptor activation. Biophysical measurements and simulations revealed that conformational “clamping” by anionic lipids promotes receptor activation (Westerfield et al., 2021). Recent work extends this principle: phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P ₂ ] organizes EphA2 together with EGFR into lipid-dependent complexes (Singh et al., 2024), while cholesterol modulates EphA2 dimerization and conformational switching (Sahoo et al., 2025). These studies and also work on the intracellular region of EphA2 (Shrestha et al., 2025) underscore the dynamic role of the membrane in shaping receptor architecture and cross talk.References: Bhattarai, N., Morrison, L., Gomes, AF., Sahoo, AR, Buck, M. (2025) Computation model predicts Rho GTPase function with the Plexin Transmembrane receptor GAP activity on Rap1b via dynamic allosteric changes Protein Science, bioRxiv https://doi.org/10.1101/2025.03.13.643120 Li, Z., Muller-Greven , J., Kim, S-J, & Buck, M. (2021) “Plexin-B1 GAP function is regulated in solution by a new inhibitory loop: Evidence for allostery in the receptor’s signaling mechanisms involving the juxtamembrane domain”. Cell. & Mol. Life Sciences 78:1101-1112. Sahoo, A., Souza, PCT., Meng,, Y., & Buck, M. (2023) “Transmembrane region dimer structures of Type 1 receptors readily sample alternate configurations: MD simulations using the Martini 3 coarse grained model compared to AlphaFold2 Multimer” Structure (Cell Press) 31:735-745.e2. Sahoo, AR., Bhattarai, N., Buck, M. (2025) Cholesterol-Dependent Dimerization and Conformational Dynamics of EphA2 Receptors: Insights from Coarse-Grained and All-Atom Simulations online at Structure 4/24/25 and online at bioRxiv https://doi.org/10.1101/2025.01.07.631553 Shrestha, P., Sahoo , A.R., Iannucci , M., Willard, , B. & Buck, M. (2025) Tyrosine phosphorylation and the inhibitory C-terminal SAM domain moderately affect transient interactions in a EphA2 cytoplasmic fragment in solution: A combined experimental and molecular modeling study. Prepublished at BIORXIV/2025/679228 and submitted to Proc.Natl.Acad.Sci. USA Singh, PK., Rybak, JA , Schuck, RJ , Sahoo, AR , Buck, M., Barrera, FN., Smith, AW. (2024) Phosphatidylinositol (4,5)- bisphosphate drives the formation of EGFR and EphA2 complexes. Science Adv. 10(49):eadl0649 Westerfield JM, Sahoo AR, Alves DS, Grau B, Cameron A, Maxwell M, Schuster JA, Souza PCT, Mingarro I, Buck M. & Barrera FN. (2021) “Conformational Clamping

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

by a Membrane Ligand Activates the EphA2 Receptor.” J Mol Biol. 433:167144. Zhang, L.*, & Buck, M.* (2017) Molecular Dynamics Simulations Reveal Isoform Specific Contact Dynamics Between the Plexin Rho GTPase Binding Domain (RBD) and Small Rho GTPases Rac1 and Rnd1. J Phys Chem B. 121:1485-1498.

SPRY2 INTERACTS WITH ACTIVE RAS TO MODULATE ITS MEMBRANE ORGANIZATION Daniel Abankwa 1 ; 1 University of Luxembourg, Department of Life Sciences and Medicine, Esch-sur-Alzette, Luxembourg K-Ras is regulated and active in nanoscale proteo-lipid domains of the plasma membrane. Only few regulators of K-Ras membrane organisation have been identified so far. We here combined a primary TurboID based proximal proteome screen with a secondary BRET-screen to identify eight novel bona fide interactors of the K-Ras G-domain that would modulate its membrane organisation. We focused our hit characterisation on one novel candidate, APLP2, and SPRY2, which was previously implicated as negative regulator of MAPK-signalling. While APLP2 appeared to indirectly bind to K-Ras via C-Raf, SPRY2 displayed characteristics of a new effector protein. We show by co-immunoprecipitation and BRET-experiments that the C terminal fragment of SPRY2 comprising residues 161-315 interacts more with K-RasG12V than the full-length protein. Both full length and C-terminal fragment localized to the membrane. SPRY2 plasma membrane localization was blocked if K-Ras membrane anchorage was inhibited. Likewise, binding to oncogenic K-Ras was disrupted by K-Ras-inhibitors. Mutations at the predicted interface of the K-Ras effector binding region and the C-terminal fragment of SPRY2 modulated the interaction. Our data further suggest that SPRY2 operates as homo- or hetero-di/oligomer with SPRY4. Both full length SPRY2 and its C-terminal fragment promote differentiation of muscle C2C12 cells, which requires inhibition of MAPK-signalling. We propose a model, wherein active K-Ras recruits SPRY-dimers via the C-terminus of SPRY2 to the plasma membrane where they bind directly to Ras and block access of effectors.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

CAPTURING LIPID-PROTEIN INTERACTIONS DURING RAS-RAF ACTIVATION USING MACHINE LEARNING-DRIVEN MULTISCALE SIMULATIONS Helgi I. Ingólfsson 1 ; 1 Lawrence Livermore National Laboratory, Livermore, CA, USA Molecular dynamics (MD) simulations offer detailed insights into biological mechanisms. However, using MD to capturing conformational transitions between different protein states can be extremely challenging, especially given complex reaction coordinates, unknown, and/or rugged energy surfaces. To address these challenges, we extended the massively parallel Multiscale Machine-Learned Modeling Infrastructure (MuMMI) by incorporating a machine learning (ML)-based structure generator. Two sets of MD simulation ensembles are executed, representing the two endpoints we want to sample protein configurations between. The ML structure generator is trained on the endpoint ensembles and a MuMMI multiscale simulation is then run, where ML-generated structures are drawn from latent space paths between the two endpoint ensembles. For each selected structure, MD simulations are performed, lipids are pre equilibrated using a macro model, and the systems are set up and run using coarse-grained simulations. These running simulations guide further structure selection, refining the sampling between the endpoints. The ML model is iteratively retrained with incoming simulation data, enhancing the latent space representations until sufficiently fine-grained paths between the two endpoint ensembles are achieved.Using the extended MuMMI protein path sampling framework we mapped the RAS-RAF protein conformations from autoinhibition to activation. The autoinhibited RAF-14-3-3_2 complex binds RAS on the plasma membrane, and multiple protein membrane engagements help loosen the autoinhibited state. After the release of RAF autoinhibition two RAF kinase domains associate forming a fully formed dimer complex.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

NOVEL TWO-STEP PERIPHERAL MEMBRANE RECRUITMENT MECHANISM OF BRUTON'S TYROSINE KINASE THROUGH NATIVE MASS SPECTROMETRY

Rachel McAllister 1,2,3 ; Kallol Gupta 2 ; Moitrayee Bhattacharyya 1 ; 1 Yale University, Department of Pharmacology, New Haven, CT, USA 2 Yale University, Department of Cell Biology, New Haven, CT, USA 3 Yale University, Nanobiology Institute, West Haven, CT, USA

Peripheral membrane proteins are critical in propagating signaling cascades from organellar membranes throughout the cell. The ability to interact with membranes dynamically in response to rapidly changing cellular conditions is absolutely crucial for their function. For many peripheral membrane proteins, membrane interactions are mediated via high-affinity specificity towards certain lipids, yet countless others form low-affinity transient interactions with other membrane lipids. These are often weak and momentary, remaining challenging to capture despite their crucial role in regulating the protein. Taking Bruton's Tyrosine Kinase (BTK), a non receptor tyrosine kinase essential for B cell maturation and activation, we demonstrate a native mass spectrometry (nMS) methodology to understand the recruitment mechanism of peripheral membrane proteins by directly studying it from lipid bilayers customized to a target organellar membrane. We demonstrate that BTK directly binds phosphatidylserine (PS) through sites distinct from those used for its activating lipid, phosphatidylinositol (3,4,5) phosphate (PIP 3 ) and that PS-bound BTK is capable of binding simultaneously to PIP 3 . Contrary to the current understanding of BTK, this suggests a PIP 3 -independent basal recruitment of BTK to the plasma membrane inner leaflet. Using auto-phosphorylation assays, we demonstrate that at physiological concentrations of PIP 3 , PS on the membrane surface enhances the amplitude of BTK activation. Our nMS and biochemical data render a model of membrane recruitment where a low-affinity interaction with abundant PS enables recruitment of BTK to the plasma membrane independent of PIP 3 . This increases the local concentration of BTK on the membrane, which, upon genesis of PIP 3 , leads to greater kinase amplification. This weak lipid-binding mediated regulation likely extends to a broader set of peripheral membrane proteins. This nMS platform can be broadly extended to further peripheral membrane proteins and diverse lipid targets – simultaneously determining tight and weak lipid binding interactions as well as their specificity and stoichiometry.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

A MINIMAL ESCRT-III SYSTEM TO MIMIC HIV-1 DETACHMENT Pulkit Aditya 1 ; Nolwenn Miguet 2 ; Winfried Weissenhorn 2 ; Patricia M. Bassereau 1 ; 1 UMR168 Institut Curie - CNRS, Physics of Cells and Cancer, Paris, France 2 UMR5075 CEA-CNRS-Université Joseph Fourier, Institut de Biologie Structurale, Grenoble, France ESCRT-III complexes mediate membrane remodeling and scission in various cellular processes. In human cells, scission typically involves a subset of 12 ESCRT-III proteins. Notably, HIV-1 budding requires only a minimal set—CHMP4B, CHMP2A, CHMP3, and the ATPase Vps4B— for viral release. Previous studies have shown that membrane scission can be inhibited and HIV 1 budding stalled when the bud neck is too wide, such as in the absence of the I-BAR protein IRSp53, even though ESCRT proteins are still recruited. Inspired by this, we reconstituted scission in vitro using the minimal human ESCRT-III machinery.Using GUVs and purified proteins, we induced membrane buds by osmotic deflation or by adding IRSp53 to generate inward tubules. In some case, CHMP4B recruitment to bud necks was enhanced via dimeric Alix. In these conditions, by fluorescence microscopy, we always observed membrane scission, even without ATP and when using C-terminally truncated CHMP4B- Δ C and CHMP2A- Δ C constructs. Scission was comparatively more efficient when adding Vps4B/ATP, in particular for the full-length proteins. In contrast, scission failed when forming a bud neck by wrapping GUV membranes around colloidal beads, even when pulling on the beads with optical tweezers, a setup previously successful using a larger set of yeast ESCRT-III. In this geometry, strong membrane-bead interactions likely constrain neck shape, preventing efficient scission.Our results suggest that the minimal ESCRT-III set used by HIV-1 can mediate scission, but with a strong dependence on membrane geometry. The broader diversity of ESCRT-III proteins in human cells may serve to ensure robust scission across a wider range of physical contexts.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

CONSTRICTING AND CLEAVING MEMBRANE NECKS WITH ESCRT-III AND VPS4 Winfried Weissenhorn 1 ; 1 University Grenoble Alpes, IBS, Grenoble, France The endosomal sorting complex for transport (ESCRT) is a highly conserved protein machinery that catalyzes a wide range of membrane remodeling processes such as vesicle formation at endosomes, budding of some enveloped viruses, membrane repair, late steps in cytokinesis and others. Common to all ESCRT-catalyzed processes is the recruitment of ESCRT-III and the ATPase VPS4, which form filaments on membranes that are remodeled and eventually disassembled by VPS4 leading to membrane constriction and cleavage. Although Eukaryotes express eleven ESCRT-III proteins called CHMP, our objectives are to demonstrate that a core complex composed of CHMP4 and/or CHMP2A and CHMP3 is sufficient to catalyze membrane fission and provide mechanistic insight into the process.We employ combined X-ray and HS AFM studies of the ESCRT-III adaptor protein Alix, which reveal that Alix dimers nucleate CHMP4B polymerization on membranes, which is likely a prerequisite for downstream polymerization of CHMP2A and CHMP3 in vivo. CryoEM structures of CHMP2A- CHMP3 copolymers ‘assembled’ within a lipid bilayer tube recapitulate the geometry of membrane neck structures generated during vesicle or virus budding and at the cytokinetic midbody. The structures demonstrate membrane interaction via electrostatic interactions and via amphipathic helices. Membrane interaction further leads to membrane thinning. Employing a ‘single’ molecule/polymer fluorescence assay and HS-AFM we show that CHMP2A-CHMP3 copolymers can constrict and cleave membranes. In conclusion, our data suggesst that CHMP2A-CHMP3 and VPS4 constitute a minimal membrane fission machinery.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

MEMBRANE INTERACTION OF A PROKARYOTIC ESCRT-III PROTEIN Lukas Schlösser 1 ; Mirka Kutzner 1 ; Benedikt Junglas 2 ; Sourav Maity 3 ; Nadja Hellmann 1 ; Wouter H Roos 3 ; Carsten Sachse 2 ; Dirk Schneider 1 ; 1 Johannes Gutenberg University, Department of Chemistry, Mainz, Germany 2 Forschungszentrum Jülich, Ernst-Ruska Centre for Microscopy and Spectroscopy with Electrons, Jülich, Germany 3 Rijksuniversiteit Groningen, Moleculaire Biofysica, Zernike Instituut, Groningen, The Netherlands The inner membrane-associated protein of 30 kDa (IM30) is involved in the biogenesis, protection and/or remodeling of internal membranes in cyanobacteria. The IM30 monomer consists of seven α -helices and spontaneously forms various homooligomeric barrel structures in solution. The oligomeric structure of IM30 exhibits a remarkable plasticity, which has also been observed with eukaryotic ESCRT-III proteins, the structural and functional homologs of IM30. In addition to barrels, we have observed the formation of rod structures in solution as well as carpets and spirals on membranes. Upon binding to solid-supported membranes, IM30 barrels disassemble into smaller oligomers, which involves partial unfolding of the monomers. In fact, the oligomeric assembly induces/stabilizes α -helical regions, and in IM30*, an IM30 variant defective in oligomerization, only a helical hairpin formed by the helices α 1-3 retains its ordered structure while the remaining regions are disordered. Membrane binding of IM30 monomers is followed by oligomerization and the formation of spiral structures on membrane surfaces, as also observed previously with eukaryotic ESCRT-III´s, as well as of barrel and rod structures. Membrane-attached IM30 barrels and rods can engulf membranes, and here membrane contacts appear to be mainly mediated by the N-terminal amphipathic helix α 0. Yet, the α 1-3 helical hairpin clearly is also involved in membrane binding in vitro and in vivo. Thus, while the N terminal helix α 0 of pro- and eukaryotic ESCRT-III´s is crucial for an interaction of ESCRT-III proteins with membranes within oligomeric assemblies, surface binding of monomeric ESCRT III subunits involves extended parts of the helix α 1-3 hairpin, the structured core conserved in all ESCRT-III superfamily members. Our findings support a conserved membrane-binding and remodeling mechanism across the ESCRT-III superfamily, shared between prokaryotic and eukaryotic proteins.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

MODELLING LIPID INTERACTIONS AT MEMBRANE INTERFACES WITH MEMPRO AND CCD2MD Phillip J. Stansfeld ; 1 University of Warwick, Coventry, United Kingdom Peripheral membrane proteins are critical regulators of membrane dynamics, often mediating curvature, lipid organization, and protein recruitment at the membrane interface. To investigate these proteins, we have developed two new computational tools specifically designed for the study of peripheral membrane interactions. MemPrO is a predictive method that identifies membrane-binding interfaces and orientations of peripheral proteins, enabling rapid screening of potential membrane-contacting regions. In parallel, we have developed CCD2MD, a flexible pipeline for setting up molecular dynamics simulations involving protein-lipid interactions and post-translational lipid modifications, using structural predictions from AlphaFold3, Chai, or Boltz. CCD2MD supports both coarse-grained and atomistic simulations, making it particularly well-suited for modeling complex membrane environments and peripheral protein behavior. As an exemplar system, we have studied the membrane interactions of MreB, a bacterial actin homolog. MreB binds the cytoplasmic leaflet of the E. coli inner membrane and remodels its architecture. Our molecular dynamics simulations show that MreB filaments recruit the cone shaped lipid cardiolipin and induce membrane bending toward the periplasmic space. This effect is cardiolipin concentration-dependent and is mediated by specific residues (R105 and R136), as well as the N-terminal amphipathic helix. These findings reveal a dual mechanism of membrane remodeling by MreB: lipid recruitment and physical distortion. The case of MreB illustrates how peripheral membrane proteins can exert both mechanical and chemical influence on membrane architecture. Our newly developed methods provide a generalizable framework for exploring similar mechanisms across a wide range of membrane-associated systems.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

PEEKING OUT FROM INSIDE THE MEMBRANE: THE CURIOUS CASE OF CAVEOLINS Anne K. Kenworthy ; 1 University of Virginia, Mol Physiol Biol Phys, Charlottesville, VA, USA Peripheral membrane proteins typically associate with one leaflet of the membrane. This characteristic is shared with monotopic membrane proteins that enter and exit the same side of the bilayer. Here, I will discuss the unusual structural features of caveolins, a family of monotopic membrane proteins located on the cytoplasmic face of the plasma membrane. Caveolins are best known for their role as building blocks of caveolae, flask-shaped invaginations that sense and relay signals and protect cells from stress. Classically, caveolins have been suggested to sit at the membrane-water interface, with large portions of the protein projecting into the cytoplasm. In contrast, our recent cryoEM structure reveals caveolins form an amphipathic disc that is predicted to be deeply embedded in the membrane. I will discuss how this unique structural organization helps shape membranes to promote caveolae assembly, impacts the ability of caveolins to serve as signaling and sensing hot spots, and serves as a conserved structural framework across the tree of life.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

MEMBRANE REMODELING BY THE COLLECTIVE ACTION OF CAVEOLIN-1 Korbinian Liebl 1,2 ; Gregory A Voth 1 ; 1 University of Chicago, Chicago, IL, USA 2 SISSA, Trieste, Italy Caveolin-1 (CAV-1) are membrane-remodeling proteins that consist of 178 amino acids. Multiple CAV-1 protomers can oligomerize into the 8S-complex that represents a disk-like structure (~14 nm diameter) with a central beta-barrel. These complexes exhibit a monotopic arrangement (inserted in only one membrane leaflet) and through higher-order interactions remodel composition and shape of the membrane, a process that is critically implicated in many cellular processes, e.g., caveolae biogenesis. The mechanistic and cooperative functioning of these CAV-1 8S complexes, however, has remained elusive. To address this shortcoming, we have performed 1µs long atomistic MD simulations that gave new insight into membrane remodeling mechanisms by this complex. For instance, we have found that the beta-barrel can store and extract cholesterol from the membrane and that post-translational modifications enhance cholesterol accumulation by the entire complex. To address the open large-scale questions and noticing that custom top-down coarse-grained (CG) models may lead to an unphysical description of this complex, we have developed a new bottom-up CG model that captures the binding of the complex in the membrane as well as relevant membrane properties correctly. In this presentation, I will present the result of the parameterization of such a CG model that facilitates significant length scaling beyond 100nm. Large-scale simulations with this model show cooperative membrane bending due to stress-amplification by proximate CAV1-8S complexes. In addition, we also monitor that distinct complexes can bind to each other, hence promoting cluster formation. We further show that such clusters scaffold ~70nm broad membrane invaginations, and additional cofactors are needed for further restriction and budding.

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Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Tuesday Speaker Abstracts

ENERGETICS OF SIDE CHAIN PARTITIONING ACROSS THE BILAYER INTERFACE Karen G. Fleming ; 1 Johns Hopkins University, Biophysics, Baltimore, MD, USA The chemical composition of the bilayer interface is complex and is characterized by a steep polarity gradient dominated by a continuously changing water concentration. The concentration of water alone changes by orders of magnitude over an the extremely small distance of ~15 to 30 Å. This environment modulates the molecular forces stabilizing the membrane interactions with proteins. We have addressed this fundamental question on protein interactions with the membrane periphery by measuring the stabilities of protein side chains across the bilayer interface using a combination of experiment and simulation. Our data shows that they “hydrophobic effect” is not one effect but rather continuously changes with the interfacial polarity gradient. we have discovered a nonpolar solvation function demonstrating an empirical correlation between the surface area of the nonpolar side chain, their free energies of interface insertion, and the local water concentration in the membrane. This new function allows side chain partitioning to be accurately estimated at any location in the bilayer. At the bilayer interface surface, we find a nonpolar hydrophobicity scale similar to the “biological”, translocon based transfer free energies, indicating that the translocon energetically mimics the bilayer interface. In contrast, at the central position of the bilayer, hydrophobicity energies are twice as stabilizing for nonpolar side chains. Together these findings can be applied to increase the accuracy of computational workflows used to identify and design membrane proteins as well as bring greater insight into our understanding of how disease-causing mutations a ff ect membrane protein folding and function.

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