Complexation of integral membrane proteins by phosphorylcholine-based amphipols

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b(6)f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols ( approximately 22 kDa) of low dispersity in length, containing approximately 30 mol% octyl side groups, approximately 35 mol% PC-groups, and approximately 35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH> or =5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.     

Bacteriorhodopsin/amphipol complexes: structural and functional properties

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, ∼2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.     

Amphipols and Photosynthetic Light-Harvesting Pigment-Protein Complexes

The trimeric light-harvesting complexes II (LHCII) of plants and green algae are pigment-protein complexes involved in light harvesting and photoprotection. Different conformational states have been proposed to be responsible for their different functions. At present, detergent-solubilized LHCII is used as a model for the “light-harvesting conformation”, whereas the “quenched conformation” is mimicked by LHCII aggregates. However, none of these conditions seem to perfectly reproduce the properties of LHCII in vivo. In addition, several monomeric LHC complexes are not fully stable in detergent. There is thus a need to find conditions that allow analyzing LHCs in vitro in stable and, hopefully, more native-like conformations. Here, we report a study of LHCII, the major antenna complex of plants, in complex with amphipols. We have trapped trimeric LHCII and monomeric Lhcb1 with either polyanionic or non-ionic amphipols and studied the effect of these polymers on the properties of the complexes. We show that, as compared to detergent solutions, amphipols have a stabilizing effect on LHCII. We also show that the average fluorescence lifetime of LHCII trapped in an anionic amphipol is ~30 % shorter than in α-dodecylmaltoside, due to the presence of a conformation with 230-ps lifetime that is not present in detergent solutions.     

Molecular Dynamics Simulations of a Membrane Protein/Amphipol Complex

Amphipathic polymers known as “amphipols” provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.     

Amphipol-Trapped ExbB–ExbD Membrane Protein Complex from Escherichia coli: A Biochemical and Structural Case Study

Nutrient import across Gram-negative bacteria’s outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB–ExbD–TonB. Having purified the ExbB₄–ExbD₂ complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB₄–ExbD₂ complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB₄–ExbD₂ was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB₄–ExbD₂ complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB₆–ExbD₄, and propose a structural arrangement of its transmembrane α-helical domains.     

Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase

Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation.     

Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.     

Effect of ceramide N-acyl chain and polar headgroup structure on the properties of ordered lipid domains (lipid rafts)

Ceramides are sphingolipids that greatly stabilize ordered membrane domains (lipid rafts), and displace cholesterol from them. Ceramide-rich rafts have been implicated in diverse biological processes. Because ceramide analogues have been useful for probing the biological function of ceramide, and may have biomedical applications, it is important to characterize how ceramide structure affects membrane properties, including lipid raft stability and composition. In this report, fluorescence quenching assays were used to evaluate the effect of analogues of ceramide with different N-acyl chains or different sphingoid backbones on raft stability and sterol content. The effect of replacing 18 mol% of sphingomyelin (SM) with ceramide in vesicles composed of a 1:1 (mol:mol) mixture of SM and dioleoylphosphatidylcholine (DOPC), with or without 25 mol% sterol, was examined. In the absence of sterol, the thermal stability of the SM-rich ordered domains increased with ceramide N-acyl chain length in the order C2:0 ∼ C6:0 ∼ C8:0 < no ceramide < C12:0 < C16:0. In vesicles containing 25 mol% cholesterol (1:1:0.66 sphingolipid:DOPC:cholesterol), the dependence of raft stability on ceramide N-acyl chain length increased in the order C8:0 ∼ C6:0 < C2:0 < C12:0 ∼ no ceramide < C16:0. We also studied the stability of lipid rafts in the presence of N-lauroyl- and N-palmitoylsphingosine analogues containing altered structures in or near the polar portion of the sphingoid base. In almost all cases, the analogues stabilized rafts to about the same degree as a normal ceramide containing the same acyl chain. The only exception was N-palmitoyl-4D-ribophytosphingosine, which was very strongly raft-stabilizing. We conclude that variations in sphingoid base structure induce only insignificant changes in raft properties. N-Lauroyl and N-palmitoylsphingosine and their analogues displaced sterol from rafts to a significant degree. Both C12:0 and C16:0 analogues of ceramide may be good mimics of natural ceramide, and useful for cellular studies in which maintenance of the normal physical properties of ceramide are important.     

Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET

The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII₁₅₇; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with K_D values (∼ 200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker (K_D  ∼ 16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer-dimer association.     

The Use of Amphipols for NMR Structural Characterization of 7-TM Proteins

While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labeled GPCRs are still very limited. Here we report on the recent progress toward NMR structural studies of the melanocortin-2 and -4 receptors, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical-scale refolding experiments on the melanocortin-2 receptor show very similar behavior to the results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straightforward and impeded detailed NMR insights for the two GPCRs. While well-resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.     

A broad glass transition in hydrated proteins

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g ∼ 180 ± 15 K. This result agrees with a broad range of T_g ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r²> at temperatures around T_D ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused.     

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V_th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V_th ∼ 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV ≤ V_th ≤ 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V_th ∼ 250 mV) followed by a noticeable destabilization (V_th ∼ 100 mV at 20%). Interestingly, the presence of a model protein, α-hemolysin, dramatically disfavours membrane poration and V_th shows a 4-fold increase (∼ 800 mV) from a protein density in the membrane of 24 × 10^− 3 proteins/μm². In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.     

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.     

SEA domain autoproteolysis accelerated by conformational strain: energetic aspects

A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G^− 1S^+ 1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N → O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t_½) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by ∼ 10 kcal mol^− 1 compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to ∼ 11 and ∼ 15 kcal mol^− 1, respectively, but ∼ 7 kcal mol^− 1 of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of ∼ 7 kcal mol^− 1 was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G^− 1A^+ 1VVV motif) and the wild-type protein. The results imply that cleavage by N → O acyl shift alone would proceed with a t_½ of ∼ 2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.     

Micelle formation in the presence of photosystem I

The correlation between membrane protein solubilisation and detergent aggregation in aqueous solution is studied for a series of n-alkyl-β-d-maltosides (C_xG₂ with x = 10, 11, 12 being the number of carbon atoms in the alkyl chain) using the trimeric photosystem I core complex (PSIcc) of oxygenic photosynthesis from Thermosynechococcus elongatus as model protein. While protein solubilisation is monitored via the turbidity of the solution, the aggregation behavior of the detergent is probed via the fluorescence spectrum of the polycyclic aromatic hydrocarbon pyrene. In addition, changes of the fluorescence spectrum of PSIcc in response to formation of the detergent belt surrounding its hydrophobic surface are investigated. Solubilisation of PSIcc and aggregation of detergent into micelles or belts are found to be strictly correlated. Both processes are complete at the critical solubilisation concentration (CSC) of the detergent, at which the belts are formed. The CSC depends on the concentration of the membrane protein, [prot], and is related to the critical micelle concentration (CMC) by the empirical law ln(CSC/CMC) = n¯₀ [prot], where the constant n¯₀ = (2.0 ± 0.3) μM⁻¹ is independent of the alkyl chain length x. Formation of protein-free micelles below the CSC is not observed even for x = 10, where a significant excess of detergent is present at the CSC. This finding indicates an influence of PSIcc on micelle formation that is independent of the binding of detergent to the hydrophobic protein surface. The role of the CSC in the optimisation of membrane protein crystallisation is discussed.     

Use of amphipathic polymers to deliver a membrane protein to lipid bilayers

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.     

Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding

We develop coarse-grained models and effective energy functions for simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (K_d > 1 μM) and apply them to binding of Vps27 to membrane-tethered ubiquitin. Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Hückel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. We parameterize the energy functions by fitting the osmotic second virial coefficient of lysozyme and the binding affinity of the ubiquitin-CUE complex. For validation, extensive replica-exchange Monte Carlo simulations are performed of various protein complexes. Binding affinities for these complexes are in good agreement with the experimental data. The simulated structures are clustered on the basis of distance matrices between two proteins and ranked according to cluster population. In ∼ 70% of the complexes, the distance root-mean-square is less than 5 Å from the experimental structures. In ∼ 90% of the complexes, the binding interfaces on both proteins are predicted correctly, and in all other cases at least one interface is correct. Transient and nonspecifically bound structures are also observed. With the validated model, we simulate the interaction between the Vps27 multiprotein complex and a membrane-tethered ubiquitin. Ubiquitin is found to bind preferentially to the two UIM domains of Vps27, but transient interactions between ubiquitin and the VHS and FYVE domains are observed as well. These specific and nonspecific interactions are found to be positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the ∼ 300 μM of the specific domains. We also find that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.     

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

The Na⁺-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 (∼ 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated (∼ 68-90 kDa) with only 31-65% of WT l-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered (∼ 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at ∼ 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 °C or 37 °C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.     

EmrE dimerization depends on membrane environment

The small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein is a homodimer in isotropic bicelles with a monomer–dimer equilibrium constant (K_MD^2D) of 0.002–0.009 mol% for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the K_MD^2D is only 0.8–0.95 mol% (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes K_MD^2D is 0.0005–0.0008 mol% based on Förster resonance energy transfer (FRET) measurements, slightly tighter than bicelles. These results emphasize the importance of the lipid membrane in influencing dimer affinity.     

Pentameric Assembly of Potassium Channel Tetramerization Domain-Containing Protein 5

We report the X-ray crystal structure of human potassium channel tetramerization domain-containing protein 5 (KCTD5), the first member of the family to be so characterized. Four findings were unexpected. First, the structure reveals assemblies of five subunits while tetramers were anticipated; pentameric stoichiometry is observed also in solution by scanning transmission electron microscopy mass analysis and analytical ultracentrifugation. Second, the same BTB (bric-a-brac, tramtrack, broad complex) domain surface mediates the assembly of five KCTD5 and four voltage-gated K ⁺ (Kv) channel subunits; four amino acid differences appear crucial. Third, KCTD5 complexes have well-defined N- and C-terminal modules separated by a flexible linker that swivels by ∼ 30^o; the C-module shows a new fold and is required to bind Golgi reassembly stacking protein 55 with ∼ 1 μM affinity, as judged by surface plasmon resonance and ultracentrifugation. Fourth, despite the homology reflected in its name, KCTD5 does not impact the operation of Kv4.2, Kv3.4, Kv2.1, or Kv1.2 channels.