Structure and inhibition mechanism of the human citrate transporter NaCT.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants.

The phosphatase FIG4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes. We therefore tested the ability of reduced CLCN7 expression to compensate for loss of FIG4 or VAC14. Knock-out of CLCN7 corrected lysosomal swelling and partially corrected lysosomal hyperacidification in FIG4 null cell cultures. Knockout of the related transporter CLCN6 (ClC-6) in FIG4 null cells did not affect the lysosome phenotype. In the Fig4 null mouse, reduction of ClC-7 by expression of the dominant negative CLCN7 variant p.Gly215Arg improved growth and neurological function and increased lifespan by 20%. These observations demonstrate a role for the CLCN7 chloride transporter in pathogenesis of FIG4 and VAC14 disorders. Reduction of CLCN7 provides a new target for treatment of FIG4 and VAC14 deficiencies that lack specific therapies, such as Charcot-Marie-Tooth Type 4J and Yunis-Varón syndrome.

Lysosomal Storage and Albinism Due to Effects of a De Novo CLCN7 Variant on Lysosomal Acidification.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl-/H+ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and -genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Diagnosis and discovery: Insights from the NIH Undiagnosed Diseases Program.

Living with an undiagnosed medical condition places a tremendous burden on patients, their families, and their healthcare providers. The Undiagnosed Diseases Program (UDP) was established at the National Institutes of Health (NIH) in 2008 with the primary goals of providing a diagnosis for patients with mysterious conditions and advancing medical knowledge about rare and common diseases. The program reviews applications from referring clinicians for cases that are considered undiagnosed despite a thorough evaluation. Those that are accepted receive clinical evaluations involving deep phenotyping and genetic testing that includes exome and genomic sequencing. Selected candidate gene variants are evaluated by collaborators using functional assays. Since its inception, the UDP has received more than 4500 applications and has completed evaluations on nearly 1300 individuals. Here we present six cases that exemplify the discovery of novel disease mechanisms, the importance of deep phenotyping for rare diseases, and how genetic diagnoses have led to appropriate treatment. The creation of the Undiagnosed Diseases Network (UDN) in 2014 has substantially increased the number of patients evaluated and allowed for greater opportunities for data sharing. Expansion to the Undiagnosed Diseases Network International (UDNI) has the possibility to extend this reach even farther. Together, networks of undiagnosed diseases programs are powerful tools to advance our knowledge of pathophysiology, accelerate accurate diagnoses, and improve patient care for patients with rare conditions.

Solvent accessibility changes in a Na+-dependent C4-dicarboxylate transporter suggest differential substrate effects in a multistep mechanism.

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site-associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.

Protons in small spaces: Discrete simulations of vesicle acidification.

The lumenal pH of an organelle is one of its defining characteristics and central to its biological function. Experiments have elucidated many of the key pH regulatory elements and how they vary from compartment-to-compartment, and continuum mathematical models have played an important role in understanding how these elements (proton pumps, counter-ion fluxes, membrane potential, buffering capacity, etc.) work together to achieve specific pH setpoints. While continuum models have proven successful in describing ion regulation at the cellular length scale, it is unknown if they are valid at the subcellular level where volumes are small, ion numbers may fluctuate wildly, and biochemical heterogeneity is large. Here, we create a discrete, stochastic (DS) model of vesicular acidification to answer this question. We used this simplified model to analyze pH measurements of isolated vesicles containing single proton pumps and compared these results to solutions from a continuum, ordinary differential equations (ODE)-based model. Both models predict similar parameter estimates for the mean proton pumping rate, membrane permeability, etc., but, as expected, the ODE model fails to report on the fluctuations in the system. The stochastic model predicts that pH fluctuations decrease during acidification, but noise analysis of single-vesicle data confirms our finding that the experimental noise is dominated by the fluorescent dye, and it reveals no insight into the true noise in the proton fluctuations. Finally, we again use the reduced DS model explore the acidification of large, lysosome-like vesicles to determine how stochastic elements, such as variations in proton-pump copy number and cycling between on and off states, impact the pH setpoint and fluctuations around this setpoint.

Characterizing chloride-dependent acidification in brain clathrin-coated vesicles 1.

Endocytic organelles maintain their acidic pH using the V-type ATPase proton pump. However, proton accumulation across the membrane generates a voltage and requires the movement of an additional ion, known as a counterion, to dissipate charge buildup. The role of counterion movement in endosomes is not clear, but a subpopulation of early endosomes, clathrin-coated vesicles (CCVs), has previously been shown to use external chloride (Cl-) to allow V-ATPase-dependent acidification. We aimed to determine the identity and function of this presumed Cl- transporting protein. Our sample of highly enriched bovine brain CCVs exhibited V-type ATPase-facilitated acidification in the presence of external Cl-, independent of the monovalent cations present. While unsuccessful at identifying the mechanism of anion transport, we used glutamate-facilitated acidification, density gradients, and mass spectrometry to show that most brain CCVs are synaptic vesicles, complementing results from earlier studies that argued similarity only on the basis on protein content. The source of Cl--dependent acidification in brain CCVs may be vGLUT1, a synaptic vesicle glutamate transporter with known Cl- permeability, although CCVs in other tissues are likely to utilize different proteins to facilitate acidification.

Lysosomal acidification mechanisms.

Lysosomes, the terminal organelles on the endocytic pathway, digest macromolecules and make their components available to the cell as nutrients. Hydrolytic enzymes specific to a wide range of targets reside within the lysosome; these enzymes are activated by the highly acidic pH (between 4.5 and 5.0) in the organelles' interior. Lysosomes generate and maintain their pH gradients by using the activity of a proton-pumping V-type ATPase, which uses metabolic energy in the form of ATP to pump protons into the lysosome lumen. Because this activity separates electric charge and generates a transmembrane voltage, another ion must move to dissipate this voltage for net pumping to occur. This so-called counterion may be either a cation (moving out of the lysosome) or an anion (moving into the lysosome). Recent data support the involvement of ClC-7, a Cl(-)/H(+) antiporter, in this process, although many open questions remain as to this transporter's involvement. Although functional results also point to a cation transporter, its molecular identity remains uncertain. Both the V-ATPase and the counterion transporter are likely to be important players in the mechanisms determining the steady-state pH of the lysosome interior. Exciting new results suggest that lysosomal pH may be dynamically regulated in some cell types.

Mechanism of transport modulation by an extracellular loop in an archaeal excitatory amino acid transporter (EAAT) homolog.

Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na(+)-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt(Ph), suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3-4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt(Ph) is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt(Ph) retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process.

The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes.

Lysosomes are the stomachs of the cell-terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5). However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.

Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT.

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.

The 3-4 loop of an archaeal glutamate transporter homolog experiences ligand-induced structural changes and is essential for transport.

Glutamatergic synaptic transmission is terminated by members of the excitatory amino acid transporter (EAAT) family of proteins that remove glutamate from the synaptic cleft by transporting it into surrounding glial cells. Recent structures of a bacterial homolog suggest that major motions within the transmembrane domain translocate the substrate across the membrane. However, the events leading to this large structural rearrangement are much less clear. Two reentrant loops have been proposed to act as extracellular and intracellular gates, but whether other regions of these proteins play a role in the transport process is unknown. We hypothesized that transport-related conformational changes could change the solvent accessibilities of affected residues, as reflected in protease sensitivity or small-molecule reactivity. In the model system Glt(Ph), an archaeal EAAT homologue from Pyrococcus horikoshii, limited trypsin proteolysis experiments initially identified a site in the long extracellular loop that stretches between helices 3 and 4 that becomes protected from proteolysis in the presence of a substrate, L-aspartate, or an inhibitor, DL-TBOA in the presence of Na(+), the cotransported ion. Using a combination of site-directed cysteine-scanning mutagenesis and fluorescein-5-maleimide labeling we found that positions throughout the loop experience these ligand-induced conformational changes. By selectively cleaving the 3-4 loop (via introduced Factor Xa sites) we demonstrate that it plays a vital role in the transport process; though structurally intact, the cleaved proteins are unable to transport aspartate. These results inculcate the 3-4 loop as an important player in the transport process, a finding not predicted by any of the available crystal structures of Glt(Ph).

The uncoupled chloride conductance of a bacterial glutamate transporter homolog.

Glutamate transporters (EAATs) are pivotal in mammalian synaptic transmission, tightly regulating synaptic levels of this excitatory neurotransmitter. In addition to coupled glutamate transport, the EAATs also show an uncoupled Cl(-) conductance, whose physiological importance has recently been demonstrated. Little is yet known about the molecular mechanism of chloride permeation. Here we show that Glt(Ph), a bacterial EAAT homolog whose structure has been determined, displays an uncoupled Cl(-) conductance that can determine the rate of substrate uptake. A mutation analogous to one known to specifically affect Cl(-) movement in EAAT1 has similar effects on Glt(Ph), suggesting that this protein is an excellent structural model for understanding Cl(-) permeation through the EAATs. We also observed an uncoupled Cl(-) conductance in another bacterial EAAT homolog but not in a homolog of the Na(+)/Cl(-)-coupled neurotransmitter transporters.

Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance.

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this 'counterion pathway' The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here, we demonstrate that depleting PI(3,5)P2 by inhibiting the kinase PIKfyve causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together, these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

Voltage-sensor activation with a tarantula toxin as cargo.

The opening and closing of voltage-activated Na+, Ca2+ and K+ (Kv) channels underlies electrical and chemical signalling throughout biology, yet the structural basis of voltage sensing is unknown. Hanatoxin is a tarantula toxin that inhibits Kv channels by binding to voltage-sensor paddles, crucial helix-turn-helix motifs within the voltage-sensing domains that are composed of S3b and S4 helices. The active surface of the toxin is amphipathic, and related toxins have been shown to partition into membranes, raising the possibility that the toxin is concentrated in the membrane and interacts only weakly and transiently with the voltage sensors. Here we examine the kinetics and state dependence of the toxin-channel interaction and the physical location of the toxin in the membrane. We find that hanatoxin forms a strong and stable complex with the voltage sensors, far outlasting fluctuations of the voltage sensors between resting (closed) conformations at negative voltages and activated (open) conformations at positive voltages. Toxin affinity is reduced by voltage-sensor activation, explaining why the toxin stabilizes the resting conformation. We also find that when hanatoxin partitions into membranes it is localized to an interfacial region, with Trp 30 positioned about 8.5 A from the centre of the bilayer. These results demonstrate that voltage-sensor paddles activate with a toxin as cargo, and suggest that the paddles traverse no more than the outer half of the bilayer during activation.

Insights into the ClC-4 transport mechanism from studies of Zn2+ inhibition.

The CLC family of chloride channels and transporters is a functionally diverse group of proteins important in a wide range of physiological processes. ClC-4 and ClC-5 are localized to endosomes and seem to play roles in the acidification of these compartments. These proteins were recently shown to function as Cl(-)/H(+) antiporters. However, relatively little is known about the detailed mechanism of CLC-mediated Cl(-)/H(+) antiport, especially for mammalian isoforms. We attempted to identify molecular tools that might be useful in probing structure-function relationships in these proteins. Here, we record currents from human ClC-4 (hClC-4) expressed in Xenopus oocytes, and find that Zn(2+) inhibits these currents, with an apparent affinity of approximately 50 microM. Although Cd(2+) has a similar effect, Co(2+) and Mn(2+) do not inhibit hClC-4 currents. In contrast, the effect of Zn(2+) on the ClC-0 channel, Zn(2+)-mediated inhibition of hClC-4 is minimally voltage-dependent, suggesting an extracellular binding site for the ion. Nine candidate external residues were tested; only mutations of three consecutive histidine residues, located in a single extracellular loop, significantly reduced the effect of Zn(2+), with one of these making a larger contribution than the other two. An analogous tri-His sequence is absent from ClC-0, suggesting a fundamentally different inhibitory mechanism for the ion on hClC-4. Manipulations that alter transport properties of hClC-4, varying permeant ions as well as mutating the "gating glutamate", dramatically affect Zn(2+) inhibition, suggesting the involvement of a heretofore unexplored part of the protein in the transport process.

The poststructural festivities begin.

ClC chloride channels orchestrate the movement of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. In this issue of Neuron, Jentsch, Pusch, and colleagues use the structure of a bacterial ClC homolog to guide a mutagenic analysis of inhibitor binding to ClC-0, ClC-1, and ClC-2.

The prokaryotic Na+/Ca2+ exchanger NCX_Mj transports Na+ and Ca2+ in a 3:1 stoichiometry.

Intracellular Ca2+ signals control a wide array of cellular processes. These signals require spatial and temporal regulation of the intracellular Ca2+ concentration, which is achieved in part by a class of ubiquitous membrane proteins known as sodium-calcium exchangers (NCXs). NCXs are secondary-active antiporters that power the translocation of Ca2+ across the cell membrane by coupling it to the flux of Na+ in the opposite direction, down an electrochemical gradient. Na+ and Ca2+ are translocated in separate steps of the antiport cycle, each of which is thought to entail a mechanism whereby ion-binding sites within the protein become alternately exposed to either side of the membrane. The prokaryotic exchanger NCX_Mj, the only member of this family with known structure, has been proposed to be a good functional and structural model of mammalian NCXs; yet our understanding of the functional properties of this protein remains incomplete. Here, we study purified NCX_Mj reconstituted into liposomes under well-controlled experimental conditions and demonstrate that this homologue indeed shares key functional features of the NCX family. Transport assays and reversal-potential measurements enable us to delineate the essential characteristics of this antiporter and establish that its ion-exchange stoichiometry is 3Na+:1Ca2+ Together with previous studies, this work confirms that NCX_Mj is a valid model system to investigate the mechanism of ion recognition and membrane transport in sodium-calcium exchangers.

Pinning Down the Mechanism of Transport: Probing the Structure and Function of Transporters Using Cysteine Cross-Linking and Site-Specific Labeling.

Transporters are crucial in a number of cellular functions, including nutrient uptake, cell signaling, and toxin removal. As such, transporters are important drug targets and their malfunction is related to several disease states. Treating transporter-related diseases and developing pharmaceuticals targeting transporters require an understanding of their mechanism. Achieving a detailed understanding of transporter mechanism depends on an integrative approach involving structural and computational approaches as well as biochemical and biophysical methodologies. Many of the elements of this toolkit exploit the unique and useful chemistry of the amino acid cysteine. Cysteine offers researchers a specific molecular handle with which to precisely modify the protein, which enables the introduction of biophysical probes to assess ligand binding and the conformational ensemble of the transporter, to topologically map transporters and validate structural models, and to assess essential conformational changes. Here, we summarize several uses for cysteine-based labeling and cross-linking in the pursuit of understanding transporter mechanism, the common cysteine-reactive reagents used to probe transporter mechanism, and strategies that can be used to confirm cysteine cross-link formation. In addition, we provide methodological considerations for each approach and a detailed procedure for the cross-linking of introduced cysteines, and a simple screening method to assess cross-link formation.