Beyond the classical ER/Golgi-dependent secretory pathway, multiple alternative mechanisms of protein secretion from cells have been discovered, processes collectively referred to as ‘unconventional protein secretion’ (UPS) (Rabouille, 2017; Dimou and Nickel, 2018; Pallotta and Nickel, 2020; Sparn et al., 2022b). The group of cargo proteins secreted by ER/Golgi-independent pathways is dominated by factors with fundamental functions in physiological processes such as inflammation and angiogenesis, among others, processes that are frequently linked to disease (Akl et al., 2016; Sitia and Rubartelli, 2018). Prominent examples for cargo proteins making use of unconventional mechanisms of protein secretion are interleukin 1β (IL1β) and fibroblast growth factor 2 (FGF2) (Nickel and Seedorf, 2008; Nickel and Rabouille, 2009; Zhang and Schekman, 2013; Rabouille, 2017; Sparn et al., 2022b). With regard to soluble cytoplasmic proteins lacking N-terminal signal peptides, two principal pathways have been identified. One of them is characterized by direct protein translocation across the plasma membrane, a process termed UPS type I with FGF2 (Sparn et al., 2022b), HIV-Tat (Rayne et al., 2010; Schatz et al., 2018), Tau (Katsinelos et al., 2018; Merezhko et al., 2018; Katsinelos et al., 2021), and homeoproteins (Amblard et al., 2020; Joliot and Prochiantz, 2022) being examples. A second pathway mediating unconventional secretion of soluble cytoplasmic cargo proteins known as UPS type III is based on intracellular vesicle intermediates such as autophagosomes or endocytic compartments (Malhotra, 2013; Zhang and Schekman, 2013; Rabouille, 2017; Dimou and Nickel, 2018; Ye, 2018; Liu et al., 2020). Interestingly, certain cargo proteins such as IL1β can be secreted via both type I and type III UPS pathways, depending on cell types and the physiological conditions that apply (Dupont et al., 2011; Liu et al., 2016; Evavold et al., 2018; Heilig et al., 2018; Monteleone et al., 2018; Chiritoiu et al., 2019; Liu et al., 2020; Pallotta and Nickel, 2020; Zhang et al., 2020). These examples reveal the complexity of secretory processes in mammalian cells as being much more diverse than previously assumed.

The unconventional secretory pathway of FGF2 is based on a small number of components, all of which are physically associated with the plasma membrane (Sparn et al., 2022b). The machinery can be classified into auxiliary factors and core machinery components. Auxiliary components are the Na,K-ATPase (Florkiewicz et al., 1998; Dahl et al., 2000; Ebert et al., 2010; Zacherl et al., 2015) and Tec kinase that binds to PI(3,4,5)P₃ at the inner plasma membrane leaflet (Ebert et al., 2010; Steringer et al., 2012; La Venuta et al., 2016), factors that mediate initial steps of this pathway. The Na,K-ATPase has been demonstrated to be the first contact of FGF2 at the plasma membrane mediated by a direct physical interaction between its α1 subunit and FGF2 (Legrand et al., 2020). It is believed to serve as a landing platform of FGF2 at the inner plasma membrane leaflet, however, it has also been hypothesized to play an additional regulatory role in coupling FGF2 membrane translocation to the maintenance of the plasma membrane potential (Lolicato and Nickel, 2022; Sparn et al., 2022b). In addition, Tec kinase has been shown to make direct physical contact with FGF2, resulting in tyrosine phosphorylation of FGF2 at Y81, an interaction that is likely to occur downstream of the Na,K-ATPase (La Venuta et al., 2015; La Venuta et al., 2016; Lolicato and Nickel, 2022; Sparn et al., 2022b). This modification has been proposed to regulate the overall efficiency of FGF2 secretion, in particular in the context of cancer development (Ebert et al., 2010; Steringer et al., 2012; La Venuta et al., 2015; La Venuta et al., 2016; Sparn et al., 2022b).

Once FGF2 is handed over from the Na,K-ATPase and Tec kinase to the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), the core mechanism of FGF2 membrane translocation into the extracellular space is triggered. It was demonstrated that the interaction with PI(4,5)P₂ leads to FGF2 oligomerization on membrane surfaces (Steringer et al., 2012; Müller et al., 2015; Steringer et al., 2017), with hexamers being the most prominent oligomeric state linked to membrane pore formation (Steringer et al., 2017; Šachl et al., 2020). In addition to the high-affinity interaction of PI(4,5)P₂ with a positively charged binding pocket containing K127, R128, and K133 (Temmerman et al., 2008; Temmerman and Nickel, 2009; Nickel, 2011; Steringer et al., 2012; Lolicato et al., 2022), molecular dynamics (MD) simulations suggest FGF2 to locally accumulate about four to five additional PI(4,5)P₂ lipids through low-affinity interactions (Steringer et al., 2017). Thus, FGF2 hexamers may accumulate up to 30 PI(4,5)P₂ lipids within a highly confined membrane surface area of about 10 nm². At these sites, given the cone-shaped structure of PI(4,5)P₂, local accumulation of PI(4,5)P₂ will likely compromise the bilayer architecture and decrease the energy barrier for the formation of toroidal membrane pores (Lolicato and Nickel, 2022; Sparn et al., 2022b). Indeed, upon PI(4,5)P₂-dependent FGF2 oligomerization on membrane surfaces, transbilayer diffusion of membrane lipids has been observed, suggesting a toroidal architecture of the membrane pores that were formed under these conditions (Steringer et al., 2012; Müller et al., 2015; Steringer et al., 2017; Dimou and Nickel, 2018; Pallotta and Nickel, 2020). With the heparan sulfate chains of glypican-1 (GPC1) containing high-affinity binding sites for FGF2 in close proximity to the membrane surface, FGF2 oligomers have been shown to get captured and disassembled at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space (Zehe et al., 2006; Nickel, 2007; Sparn et al., 2022a). This step is unidirectional as heparan sulfate chains compete with high affinity against PI(4,5)P₂ for the same binding site on the molecular surface of FGF2 (Steringer et al., 2017). Thus, in conclusion, the core machinery of FGF2 membrane translocation consists of PI(4,5)P₂ and heparan sulfate chains linked to GPC1 on opposing sites of the plasma membrane. Along with this, FGF2 locally accumulates PI(4,5)P₂ molecules through oligomerization concomitant with the formation of a membrane pore with a toroidal architecture (Pallotta and Nickel, 2020; Lolicato and Nickel, 2022; Sparn et al., 2022b).

The goal of the current study was to shed light on the structural principles that govern PI(4,5)P₂-dependent oligomerization of FGF2 on membrane surfaces. Particular emphasis was given to the role of intermolecular disulfide bridges known to be present in PI(4,5)P₂-triggered FGF2 oligomers. Furthermore, on a functional basis, it was known that a variant form of FGF2 lacking both of the two surface cysteine residues C77 and C95 is incapable of both oligomerization, membrane pore formation, and secretion from cells (Müller et al., 2015; Steringer et al., 2017; Dimou and Nickel, 2018; Steringer and Nickel, 2018). However, the way these cysteines contribute to disulfide bridge formation and their specific roles in a cellular context remained unknown. In the current study, through a combination of biochemical, cell biological, and structural techniques including cryo-electron tomography (cryo-ET), cross-linking mass spectrometry (XL-MS), atom-scale biomolecular simulations, and deep learning, we mechanistically dissected the functional roles of C77 and C95 in the sequence of events that constitute the unconventional secretory pathway of FGF2. As opposed to previous concepts, we found C77 not to be involved in FGF2 oligomerization. Rather, along with K54 and K60 (Legrand et al., 2020), we revealed a role for C77 in building the molecular interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the starting point of this unusual pathway of protein secretion. By contrast, through the formation of disulfide bridges, we found C95 to be essential for PI(4,5)P₂-dependent FGF2 oligomerization on membrane surface, producing the dimeric building blocks for higher FGF2 oligomers that can trigger the formation of lipidic membrane pores. Our findings further imply that the heparan sulfate chains of GPC1 containing high-affinity binding sites for FGF2 reverse this process by disassembling membrane pore-forming FGF2 oligomers into C95-C95 disulfide-bridged dimers. The latter are likely to represent the primary ligands for the formation of FGF2 signaling complexes for autocrine and paracrine FGF signal transmission into cells (Decker et al., 2016; Nawrocka et al., 2020). We, therefore, propose unconventional secretion of FGF2 from cells and autocrine signal transmission into cells to represent tightly coupled processes.

The principal machinery and basic aspects of the molecular mechanism by which FGF2 can physically traverse the plasma membrane to get access to the extracellular space have been revealed in great detail in recent years (Dimou and Nickel, 2018; Pallotta and Nickel, 2020; Sparn et al., 2022b). However, the mechanism by which FGF2 oligomerizes in a PI(4,5)P₂-dependent manner on membrane surfaces concomitant with the formation of a lipidic membrane pore continued to be a mystery. In particular, the molecular events remained elusive by which the highly dynamic process of PI(4,5)P₂-dependent FGF2 oligomerization triggers the remodeling of the rather stable plasma membrane lipid bilayer into a lipidic membrane pore with a toroidal architecture, the intermediate structure through which FGF2 oligomers can move toward the extracellular space. This process concludes in a GPC1-dependent manner, a cell surface heparan sulfate proteoglycan that captures FGF2 oligomers as they penetrate PI(4,5)P₂-dependent membrane pores, followed by disassembly into FGF2 signaling modules on cell surfaces (Zehe et al., 2006; Nickel, 2007; Sparn et al., 2022a). To address the long-term goal of a mechanistic understanding of how FGF2 oligomers can trigger the biogenesis of lipidic membrane pores in a PI(4,5)P₂-dependent manner, it is crucial to understand how FGF2 molecules assemble into oligomers at the inner leaflet of plasma membranes.

In previous work, we identified two surface cysteines in positions 77 and 95, respectively, that are fully conserved among all mammalian forms of FGF2 (Müller et al., 2015; Steringer et al., 2017). Of note, these cysteines cannot be found in FGF family members containing N-terminal signal peptides for ER/Golgi-dependent secretion, suggesting a specialized role in unconventional secretion of FGF2 (Steringer and Nickel, 2018). Manipulation of these cysteine residues by either NEM-mediated alkylation or substitution by alanines resulted in the inability of FGF2 to oligomerize on membrane surfaces in a PI(4,5)P₂-dependent manner (Müller et al., 2015). Such conditions further caused a failure of FGF2 to form membrane pores, to translocate across membranes and to get secreted from cells (Müller et al., 2015; Steringer et al., 2017). When oligomeric species of FGF2 were analyzed by native gel electrophoresis or by comparing migration behavior by reducing and non-reducing sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), evidence could be collected that intermolecular disulfide bridges play a role in PI(4,5)P₂-dependent FGF2 oligomerization on membrane surfaces (Müller et al., 2015). However, it remained unclear as to whether both cysteines are involved and how disulfide bridges are arranged to form FGF2 dimers and higher oligomers.

With the current study, we now obtained key insights into the structural foundations of how FGF2 dimerizes on membrane surfaces. In conjunction with functional in vitro studies and cell-based experiments, this became possible only through a combination of cryo-ET, atomistic MD simulations, machine learning analyses, structural predictions from AlphaFold2-Multimer, and insights from cross-linking mass spectrometry experiments, providing a dataset suited to solve this challenging problem. In particular, we revealed an FGF2 dimerization interface that contains a disulfide bridge that depends on C95, one of two cysteine residues on the molecular surface of FGF2. Of note, disulfide bridge formation driving FGF2 oligomerization was found to be completely independent of C77, a second residue with a thiol side chain that is localized nearby C95 on the molecular surface of FGF2. Intriguingly, our data from machine learning analysis (Figure 8), atomistic MD simulations (Figure 9), and XL-MS (Figure 10) indicate that this dimerization interface assembles independently of C95-C95 disulfide formation, meaning the stability of this configuration is not derived solely from the formation of the disulfide bridge. Instead, it offers kinetic stability essential for disulfide bridge formation at the inner leaflet of the plasma membrane, indicating that the formation of disulfide-bridged FGF2 dimers plays a critical role in stabilizing the dimerization interface during FGF2 membrane translocation. This, in turn, explains why C95 substitutions by alanine or serine cause severe FGF2 secretion phenotypes. These findings clarify the molecular configuration by which disulfide bridges are formed in membrane-associated FGF2 dimers. Furthermore, the inability of C77 to form disulfide bridges had important implications of the overall structure of higher FGF2 oligomers. In particular, we propose a second non-covalent FGF2-FGF2 interaction interface to exist, resulting in the formation of higher FGF2 oligomers in which disulfide bridges and non-covalent FGF2-FGF2 interaction interfaces occur in an alternating arrangement. The C95-dependent disulfide-mediated interface could be revealed in this study (Figures 8—11). The second disulfide-indendent interface is likely to be represented by the one identified in this study through XL-MS experiments (Figure 10A and C). The combination of these interfaces will be key for future studies, revealing the structure-function relationship of higher FGF2 oligomers forming lipidic membrane pores during FGF2 translocation into the extracellular space.

The limitation to just one type of disulfide bridge in FGF2 oligomers also has implications on how these oligomers are disassembled on cell surfaces, mediated by GPC1 (Sparn et al., 2022a; Sparn et al., 2022b). We propose the product of this process to be C95-C95-linked FGF2 dimers, generated by the GPC1-dependent disruption of the predicted second non-covalent FGF2-FGF2 interface. Of note, recombinant forms of C95-C95 FGF2 dimers stabilized by chemical cross-linking have been demonstrated to be efficient FGF2 signaling modules (Decker et al., 2016; Nawrocka et al., 2020). These observations led us to hypothesize that the unconventional secretory pathway of FGF2 and the generation of FGF2 signaling units are tightly coupled processes, with the dimeric FGF2 signaling modules that drive FGF2 signaling already forming at the inner plasma membrane leaflet. In this concept, FGF2 dimers transiently oligomerize into higher oligomers as intermediates of FGF2 membrane translocation, a process that has been shown to occur within a time interval of just 200 ms (Dimou et al., 2019). By the action of GPC1, we propose membrane-inserted FGF2 oligomers to be reconverted into FGF2 dimers at the outer leaflet, the principal units of FGF2 signal transduction (Plotnikov et al., 1999; Plotnikov et al., 2000; Schlessinger et al., 2000).

Starting from the original observation of PI(4,5)P₂-dependent oligomerization to involve intermolecular disulfide bridges (Müller et al., 2015), the current study resolved the amino acid code of this phenomenon, demonstrating an exclusive role for C95. Nevertheless, as revealed in this study, a substitution of C77 to alanine caused FGF2 secretion from cells to become less efficient (Figure 1). This phenomenon could be explained with the proximity of C77 to K54 and K60 on the molecular surface of FGF2, residues that previously have been demonstrated to be part of the molecular interaction interface between FGF2 and the α1 subunit of the Na,K-ATPase (Legrand et al., 2020). Indeed, using BLI to study the kinetics of FGF2/α1 interactions, we revealed a C77A substitution alone to severely weaken this interface. Triple substitutions (K54/60E-C77A) caused a complete failure of FGF2 to bind to the α1 subunit of the Na,K-ATPase. With these findings, we could resolve a long-standing question regarding the molecular mechanism of FGF2 membrane translocation, the differential roles of C77 and C95 with regard to FGF2 interactions with α1 and disulfide-mediated dimerization of FGF2 on membrane surfaces. Our observations have important implications for future studies revealing the mechanisms by which higher FGF2 oligomers can trigger and become accommodated in lipidic membrane pores, based on the flexible hinge produced by the C95-C95 disulfide bridge as predicted by MD simulations in this study.

It is important to note that only a few examples exist for the formation of disulfide bridges in soluble protein complexes that reside in the cytoplasm, an observation that has been attributed to the reducing properties of this compartment. For instance, viruses maturing in the cytoplasm can form stable structural disulfide bonds in their coat proteins (Locker and Griffiths, 1999; Hakim and Fass, 2010). Moreover, a number of cytosolic proteins, including phosphatases, kinases, and transcriptions factors, have been recognized to be regulated by thiol oxidation and disulfide bond formation, formed as a post-translational modification (Lennicke and Cochemé, 2021). In numerous cases with direct relevance for our studies on FGF2, disulfide bond formation and other forms of thiol oxidation occur in association with membrane surfaces. In fact, many of these processes are linked to the inner plasma membrane leaflet (Nordzieke and Medraño-Fernandez, 2018). Growth factors, hormones, and antigen receptors are observed to activate transmembrane NADPH oxidases generating O₂^·-/H₂O₂ (Brown and Griendling, 2009). For example, the local and transient oxidative inactivation of membrane-associated phosphatases (e.g. PTEN) serves to enhance receptor associated kinase signaling (Netto and Machado, 2022). It is therefore conceivable that similar processes introduce disulfide bridges into FGF2 while assembling into oligomers at the inner plasma membrane leaflet, providing a mechanism that ensures that FGF2 monomers do not prematurely oligomerize in the cytoplasm of cells. In future experiments, it will be important to study disulfide-dependent FGF2 oligomerization in a cellular context, revealing the oxidant and the enzymatic systems that mediate this process at a time scale compatible with the kinetics observed for FGF2 membrane translocation in intact cells (Dimou et al., 2019).

In conclusion, with the current study, important insights were obtained regarding the highly dynamic process of PI(4,5)P₂-dependent FGF2 oligomerization on membrane surfaces, the key trigger of the central step of the unconventional secretory pathway of FGF2, the remodeling of the plasma membrane lipid bilayer into a lipidic membrane pore with a toroidal architecture. With a number of other cargo proteins secreted by UPS type I pathways in a PI(4,5)P₂-dependent manner such as HIV-Tat, Tau, and homeoproteins (Rayne et al., 2010; Debaisieux et al., 2012; Rabouille, 2017; Katsinelos et al., 2018; Merezhko et al., 2018; Merezhko et al., 2020; Joliot and Prochiantz, 2022; Sparn et al., 2022b), our findings pave the way for a deeper understanding of the general principles of UPS type I pathways of UPS.

All structures generated using AlphaFold2-multimer (v3), as well as all initial and configuration molecular dynamics simulations files related to this study, have been deposited to ZENODO. The machine learning code to analyze the MD simulation data has been deposited to GitLab (copy archived at Kaptan and Vattulainen, 2023). The source data underpinning the plots presented in this study are available for download as supplementary Excel files.

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Author details

1. Fabio Lolicato

   1. Heidelberg University Biochemistry Center, Heidelberg, Germany
   2. Department of Physics, University of Helsinki, Helsinki, Finland

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Julia P Steringer

   For correspondence

   fabio.lolicato@bzh.uni-heidelberg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7537-0549

2. Julia P Steringer

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   Conceptualization, Resources, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Fabio Lolicato

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9418-2762

3. Roberto Saleppico

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   Conceptualization, Resources, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0502-192X

4. Daniel Beyer

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. Jaime Fernandez-Sobaberas

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   Resources, Visualization

   Competing interests

   No competing interests declared

6. Sebastian Unger

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Steffen Klein

   Schaller Research Group, Department of Infectious Diseases-Virology, Heidelberg University Hospital, Heidelberg, Germany

   Contribution

   Resources

   Competing interests

   No competing interests declared

8. Petra Riegerová

   J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Prague, Czech Republic

   Contribution

   Conceptualization, Resources

   Competing interests

   No competing interests declared

9. Sabine Wegehingel

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   Conceptualization, Resources

   Competing interests

   No competing interests declared

10. Hans-Michael Müller

    Heidelberg University Biochemistry Center, Heidelberg, Germany

    Contribution

    Conceptualization, Resources

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2384-7285

11. Xiao J Schmitt

    Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany

    Contribution

    Conceptualization, Resources

    Competing interests

    No competing interests declared

12. Shreyas Kaptan

    Department of Physics, University of Helsinki, Helsinki, Finland

    Contribution

    Conceptualization, Software, Resources, Funding acquisition, Validation, Visualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

13. Christian Freund

    Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany

    Contribution

    Conceptualization, Supervision, Funding acquisition, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

14. Martin Hof

    J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Prague, Czech Republic

    Contribution

    Supervision, Funding acquisition, Methodology

    Competing interests

    No competing interests declared

15. Radek Šachl

    J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Prague, Czech Republic

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology

    Competing interests

    No competing interests declared

16. Petr Chlanda

    Schaller Research Group, Department of Infectious Diseases-Virology, Heidelberg University Hospital, Heidelberg, Germany

    Contribution

    Conceptualization, Resources, Supervision, Validation, Visualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7782-2139

17. Ilpo Vattulainen

    Department of Physics, University of Helsinki, Helsinki, Finland

    Contribution

    Data curation, Formal analysis, Funding acquisition, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7408-3214

18. Walter Nickel

    Heidelberg University Biochemistry Center, Heidelberg, Germany

    Contribution

    Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

    For correspondence

    walter.nickel@bzh.uni-heidelberg.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-6496-8286

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