Further insights from structural mass spectrometry into endocytosis adaptor protein assemblies

As a fundament in many biologically relevant processes, endocytosis in its different guises has been arousing interest for decades and still does so. This is true for the actual transport and its initiation alike. In clathrin-mediated endocytosis, a comparatively well understood endocytic pathway, a set of adaptor proteins bind specific lipids in the plasma membrane, subsequently assemble and thus form a crucial bridge from clathrin to actin for the ongoing process. These adaptor proteins are highly interesting themselves and the subject of this manuscript. Using many of the instruments that are available now in the mass spectrometry toolbox, we added some facets to the picture of how these minimal assemblies may look, how they form, and what influences the structure. Especially, lipids in the adaptor protein complexes result in reduced charging of a normal sized complex due to their specific binding position. The results further support our structural model of a double ring structure with interfacial lipids.

Adapted from [2]. C: Aligned tertiary structures of yeast ENTH1 (PMID 5onf, blue) and ENTH2 (PMID cyan, yellow) as well as of Chaetomium thermophilum ANTH (homologous to the S.cerevisiae protein, PMID 5oo7, green). D: SAXS model of human ENTH homo-hexamer [2] with two protein subunits colored cyan, orange and grey, each and their N-terminal ten amino acids emphasized in blue, red and black, respectively. Green spheres represent C-terminal dummy residues; yellow ones the C-atoms of bound dioctanoyl l-α-phosphatidyl-d-myo-inositol 4,5-diphosphate (PIP2). To facilitate discrimination of the six different PIP2, three of them are shown with hydrogens present, the other three with hydrogens deliberately removed. Labels specify C-termini (Ct) and Nterminal α-helices (Nα) of individual subunits indicated by Arabian numbers. Larger font size and respectively darker colors of the backbone indicate N-terminal α-helices pointing out of the paper plane. Figure S2Ligand binding and complex formation of yeast ENTH1 and ANTH. Time-of Flight (ToF) mass spectra ofyeast ENTH1 (A), yeast ANTH (B) and a 1:1 mixture of both (C), all in the presence of 6x PIP2 excess. Grey peaks denote cytochrome C (A) and carbonic anhydrase (B) used as reference proteins for unspecific PIP2. Grey peaks denote cytochrome C (A) and carbonic anhydrase (B) used as reference proteins for unspecific PIP2 clustering (blue peaks) during the ESI process [4]. Light and dark green as well as orange and red peaks specify adaptor proteins in free and lipid-bound state, respectively.
The inset shows an enlarged upper part of the spectrum with blue and red lines indicating the theoretical peak positions of the di-hexamer (green) and of the di-octamer (cyan) complexes with 19 and 26 PIP2 ligands, respectively. Data taken from [2].

Figure S3
Part of native mass spectra showingcomplex formation of yeast ENTH1-ANTH di-hexa-and di-octamers.Mass spectra of yeast ENTH1 and ANTH were recorded in a Q-ToF1 mass spectrometer modified for native MS after variedsample preparations. A: ENTH1 and ANTH were first buffer-exchanged and then assembled with an excess of PIP2 (5.5 µM, 8 µM, and 30 µM, respectively) B: the complex was first assembled (same concentrations as above) and then buffer-exchanged, i.e. the excess PIP2 was removed from the mixture. Measurements were conducted identically (1.35 kV, 150 V, and 75 V capillary, cone and collision cell voltages, respectively). The spectrum shown in Bwas taken from [3].

Figure S7 Charge state dependence of individual collision cross sections (CCS').
Calculated CCS' of yeast nonameric (6:3) (I) di-hexameric (II) and di-octameric (III) ENTH2-ANTH or ENTH1-ANTH (IV) complexes as well as of the human ENTH hexamer (V) are plotted versus the corresponding charge state and in comparison with similar data of the indicated reference proteins. Data of cytochrome C (cyt C), β-lactoglobulin (β-lac) transthyretin (TT), avidin (avi), serum albumin (BSA), concanavalin A (ConA), alcohol dehydrogenase (ADH), serum amyloid P (SA P), pyruvate kinase (PK), glutamate dehydrogenase (GDH) and GroEL were taken from [5]. If deemed necessary, oligomerization status indicated by numbers in in brackets, Roman numbering refers to Tables 1 and 2 in the main section.

Figure S8 Native Ion Mobility Mass Spectrum of human ENTH in different association states. A:
Precursor ion mass spectrum comprising free PIP2 with fragments thereof and sodium adducts (I), monomeric free ENTH (II), a mixture of PIP2bound monomeric and dimeric (both, free and lipid-bound) ENTH (III) and hexameric ENTH (IV). Vertical lines indicate theoretical m/z free monomeric (green), free (cyan) or doubly ligand-bound (blue), and hexameric (red) ENTH. B: Heat map combining MS (horizontal) and ion mobility (vertical axis) dimensions. The intensity scales is logarithmic. C: Enlarged lower part of the spectrum shown in A, with the theoretical m/z of free, singly or doubly PIP2-bound monomericENTH denoted by orange, red, and dark red lines, respectively, as well as those of free, singly or doubly PIP2-bound ENTH dimers indicated by cyan, dark blue, and grey ones. *: unidentified species. See Figure S9B