Studies with chimeras of the gonadotropin receptors reveal the importance of third intracellular loop threonines on the formation of the receptor/nonvisual arrestin complex

Using chimeras and more discrete exchange mutations of the rat (r) and human (h) gonadotropin receptors, we had previously identified multiple noncontiguous residues of the lutropin (LHR) and follitropin (FSHR) receptors that dictate their rates of internalization. Since the internalization of the LHR and the FSHR is driven by their abilities to associate with the nonvisual arrestins, we hypothesized that one or more of the residues previously identified by the internalization assays are involved in the formation of the receptor/nonvisual arrestin complex. In the studies reported herein, we tested this hypothesis by measuring the association of arrestin-3 with a large number of rLHR/hLHR and rFSHR/hFSHR exchange mutants that affect internalization. The results presented show that the same residues that dictate the rate of internalization of these two receptor pairs affect their ability to associate with arrestin-3. Although these residues are located in distinct topological domains, our analyses show that threonine residues in the third intracellular loop of both receptor pairs are particularly important for the formation of the receptor/arrestin-3 complexes and internalization. We conclude that the different rates of internalization of the gonadotropin receptors are dictated by their different abilities to associate with the nonvisual arrestins and that this association is, in turn, largely dictated by the presence of threonine residues in their third intracellular loops.     

Role of arginine and histidine residues in the biological activity of botulinic neurotoxin A

Treatment of botulinic neurotoxin A with cyclohexanedione demonstrated that modification of 5 to 10 arginine residues does not change the neurotoxin toxicity, while after modification of 15-20 arginine residues the toxicity is decreased by 40-50% of the original value. Butanedione exerts a stronger detoxicating effect on neurotoxin than cyclohexanedione. The molecular conformation of the modified toxin derivatives and their precipitability upon interaction with antisera against toxin and toxin fragments does not change thereby. The non-toxic derivatives of toxin containing 40 modified arginine residues possess a partial serological affinity for the original toxin in a reaction with antiserum against toxin but do not interact with the antifragment sera. The molecular conformation of these preparations is changed considerably. It is assumed that one or two arginine residues are located near the toxic site of the neurotoxin molecule and are also components of its antigenic determinants. Modification of histidine residues in the neurotoxin molecule by diethylpyrocarbonate is accompanied by a decrease of its toxicity. An additional 10% toxicity is revealed upon modification of 11-13 histidine residues. The molecular conformation of the modified derivatives of neurotoxin and their precipitability do not change thereby. It is probable that 1 or 2 histidine residues are located at or near the toxic site. The data obtained suggest that histidine residues are not localized in antigenic determinants of the neurotoxin molecule.     

Global analysis of tandem aromatic octapeptide repeats: the significance of the aromatic-glycine motif

MOTIVATION: Tandem peptide repeats play a key role in self-assembly and aggregation processes. A notable example is the occurrence of tandem peptide repeats in prionic proteins and their role in the aggregation process that leads to the formation of the prion. One of the structural characteristics that is evident from the comparison of mammalian and yeast prion proteins is the presence of aromatic residues in their tandem repeats. These residues are accompanied by glycine residues before and/or after the aromatic amino acid. Such aromatic-glycine conjugates are also present in the tandem repeats of the large family of the bacterial ice nucleation proteins. To study the significance of such aromatic-glycine occurrences, a global analysis of all the aromatic octapeptide repeats in the Swiss-Prot and TrEMBL databases was conducted. The search pattern was formulated to compare the number of conjugates of each of the 20 natural amino acids before or after the different aromatic residues. RESULTS: The presence of aromatic-glycine conjugates appears to be significantly higher than aromatic conjugates to any other amino acid. Furthermore, all the six various combination of glycine occurrences before or after the three aromatic residues are present. No such pattern was observed for any other amino acid. The significance of the findings is being discussed in the context of the physicochemical properties of aromatic-glycine conjugates and its possible role in the facilitation of aggregates formation.     

Protein-derived cofactors. Expanding the scope of post-translational modifications

Recent advances in enzymology, structural biology, and protein chemistry have extended the scope of the field of cofactor-dependent enzyme catalysis. It has been documented that catalytic and redox-active prosthetic groups may be derived from post-translational modification of amino acid residues of proteins. These protein-derived cofactors typically arise from the oxygenation of aromatic residues, covalent cross-linking of amino acid residues, or cyclization or cleavage of internal amino acid residues. In some cases, the post-translation modification is a self-processing event, whereas in others, another processing enzyme is required. The characterization of protein-derived cofactors and their mechanisms of biogenesis introduce a new dimension to our current views about protein evolution and protein structure-function relationships.     

Acidic residues on the voltage-sensor domain determine the activation of the NaChBac sodium channel

The voltage-sensing domain of voltage-gated ion channels is characterized by specific, conserved, charged residues. Positively charged residues on segment S4 are the main contributors to voltage-sensing and negatively charged residues on the S2 and S3 segments are believed to participate to the process. However, their function in the voltage sensor is not well understood. To probe the role of three acidic residues in NaChBac (D-58 and E-68 in S2, and D-91 in S3), we employed site-directed mutagenesis to substitute native acidic residues with cysteine (neutral), lysine (positive charge), or either aspartate or glutamate (negative charge). We used a combination of the patch-clamp technique to record Na+ currents and molecular modeling to visualize interacting amino acid residues. We suggest that the acidic residues on the S2 and S3 segments form specific interactions with adjacent amino acids in the voltage-sensor domain. The main interactions in NaChBac are D-58 (S2) with A-97-G-98 (S3) and R-120 (S4), E-68 (S2) with R-129 (L4-5), and D-91 (S3) with R-72 (S2). Changing these acidic residues modified the interactions, which in turn altered the sensitivity of the voltage sensor.     

Studies of the histidine residues of human and bovine glycoprotein hormones by nuclear magnetic resonance

Titration curves of the histidine residues in lutropin, thyrotropin, follitropin and chorionic gonadotropin have been assigned using imidazole C-2 proton nuclear magnetic resonance spectra and their estimated pK values determined. Spectra of reassociated hormone preparations, in which one or the other of their two subunits (alpha or beta) have had their accessible histidines exchanged with deuterium, permitted assignment of C-2 resonance to specific residues. Similar titration curves were found for residues which are conserved from one hormone to another. However, these conserved histidines do not have identical pK values, indicating that differences in the conformation or microenvironment around these residues occur in these hormones. Changes in some pK values also occur as a function of subunit association. The most dramatic change seen in all cases is the exposure to solvent of histidine alpha-83; in isolated alpha subunits this residue is unavailable for titration over a wide pH range. This change appears to be a general consequence of the association of the two subunits in any of these hormones. The data show that all histidines in the intact hormones are accessible to the environment, including those proposed to be in domains involved in subunit-subunit interaction.     

Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the omega site) and Y or N at the site 2 amino acids upstream of the omega site for cell wall localization and (ii) dibasic residues in the region upstream of the omega site (the omega-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the omega-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.     

Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida

Active sites may be regarded as layers of residues, whereby the residues that interact directly with substrate also interact with residues in a second shell and these in turn interact with residues in a third shell. These residues in the second and third layers may have distinct roles in maintaining the essential chemical properties of the first-shell catalytic residues, particularly their spatial arrangement relative to the substrate binding pocket, and their electrostatic and dynamic properties. The extent to which these remote residues participate in catalysis and precisely how they affect first-shell residues remains unexplored. To improve our understanding of the roles of second- and third-shell residues in catalysis, we used THEMATICS to identify residues in the second and third shells of the Co-type nitrile hydratase from Pseudomonas putida (ppNHase) that may be important for catalysis. Five of these predicted residues, and three additional, conserved residues that were not predicted, have been conservatively mutated, and their effects have been studied both kinetically and structurally. The eight residues have no direct contact with the active site metal ion or bound substrate. These results demonstrate that three of the predicted second-shell residues (α-Asp164, β-Glu56, and β-His147) and one predicted third-shell residue (β-His71) have significant effects on the catalytic efficiency of the enzyme. One of the predicted residues (α-Glu168) and the three residues not predicted (α-Arg170, α-Tyr171, and β-Tyr215) do not have any significant effects on the catalytic efficiency of the enzyme.     

Assessing the role of tryptophan residues in the binding site

Instead of looking at the interfacial area as a measure of the extent of a protein--protein recognition site, a new procedure has been developed to identify the importance of a specific residue, namely tryptophan, in the binding process. Trp residues which contribute more towards the free energy of binding have their accessible surface area reduced, on complex formation, for both the main-chain and side-chain atoms, whereas for the less important residues the reduction is restricted only to the aromatic ring of the side chain. The two categories of residues are also distinguished by the presence or absence of hydrogen bonds involving the Trp residue in the complex. A comparison of the observed change in the accessible surface area with the value calculated using an analytical expression provides another way of characterizing the Trp residues critical for binding and this has been used to identify such residues involved in binding non-proteinaceous molecules in protein structures.     

Satisfaction of hydrogen-bonding potential influences the conservation of polar sidechains

Although polar amino acids tend to be found on the surface of proteins due to their hydrophilic nature, their important roles within the core of proteins are now becoming better recognized. It has long been understood that a significant number of mainchain functions will not achieve hydrogen bond satisfaction through the formation of secondary structures; in these circumstances, it is generally buried polar residues that provide hydrogen bond satisfaction. Here, we describe an analysis of the hydrogen-bonding of polar amino acids in a set of structurally aligned protein families. This allows us not only to calculate the conservation of each polar residue but also to assess whether conservation is correlated with the hydrogen-bonding potential of polar sidechains. We show that those polar sidechains whose hydrogen-bonding potential is satisfied tend to be more conserved than their unsatisfied or nonhydrogen-bonded counterparts, particularly when buried. Interestingly, these buried and satisfied polar residues are significantly more conserved than buried hydrophobic residues. Forming hydrogen bonds to mainchain amide atoms also influences conservation, with those satisfied buried polar residues that form two hydrogen bonds to mainchain amides being significantly more conserved than those that form only one or none. These results indicate that buried polar residues whose hydrogen-bonding potential is satisfied are likely to have important roles in maintaining protein structure.     

Sequence conservation provides the best prediction of the role of proline residues in p13suc1

The unique nature of the proline side-chain imposes severe constraints on the polypeptide backbone, and thus it seems likely that it plays a special structural or functional role in the architecture of proteins. We have investigated the role of proline residues in suc1, a member of the cyclin-dependent kinase (cks) family of proteins, whose known function is to bind to and regulate the activity of the major mitotic cdk. The effect on stability of mutation to alanine of all but two of the eight proline residues is correlated with their conservation within the family. The remaining two proline residues are located in the hinge loop between two beta-strands that mediates a domain-swapping process involving exchange of a beta-strand between two monomers to form a dimer pair. Mutation of these proline residues to alanine stabilises the protein. cdk binding is unaffected by these mutations, but dimerisation is altered. We propose, therefore, that the double-proline motif is conserved for the purpose of domain swapping, which suggests that this phenomenon plays a role in the function of cks proteins. Thus, the conservation of the proline residues is a good indicator of their roles in suc1, either in the stabilisation of the native state or in performing functions that are as yet unknown. In addition, the strain resulting from two of the proline residues was relieved successfully by mutation of the preceeding residue to glycine, suggesting a general method for designing more stable proteins.     

How can the aromatic side-chains modulate the conductance of the gramicidin channel? A new approach using non-coded amino acids.

In order to elucidate the role of the aromatic side-chains in the mechanism of transduction of monovalent cations through the channel of linear gramicidin, two series of analogues containing non-coded aromatic amino acids were synthesized. In the first series, the four tryptophans were replaced by either four L-3-(8-quinolyl)alanyl or four L-3-(4-quinolyl)alanyl residues and single channel conductance measurements showed that these substitutions led to a strong lowering of the channel conductance, which is attributed to a modification of the orientation of the aromatic side-chains due to an increase of their hydrophobicity. In the second series, the analogues contained both tryptophyl and naphthylalanyl residues in various amounts and positions. The single channel conductance data indicated that the conductance was mainly governed by the number of polar residues (Trp) and not by their positions. The conformational consequences of these results are discussed together with their influence on the energy profile of the gramicidin channel.     

Flexible linkers leash the substrate binding domain of SspB to a peptide module that stabilizes delivery complexes with the AAA+ ClpXP protease

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.     

Canonical structures for the hypervariable regions of immunoglobulins

We have analysed the atomic structures of Fab and VL fragments of immunoglobulins to determine the relationship between their amino acid sequences and the three-dimensional structures of their antigen binding sites. We identify the relatively few residues that, through their packing, hydrogen bonding or the ability to assume unusual phi, psi or omega conformations, are primarily responsible for the main-chain conformations of the hypervariable regions. These residues are found to occur at sites within the hypervariable regions and in the conserved beta-sheet framework. Examination of the sequences of immunoglobulins of unknown structure shows that many have hypervariable regions that are similar in size to one of the known structures and contain identical residues at the sites responsible for the observed conformation. This implies that these hypervariable regions have conformations close to those in the known structures. For five of the hypervariable regions, the repertoire of conformations appears to be limited to a relatively small number of discrete structural classes. We call the commonly occurring main-chain conformations of the hypervariable regions "canonical structures". The accuracy of the analysis is being tested and refined by the prediction of immunoglobulin structures prior to their experimental determination.     

Intrinsic disorder in the Protein Data Bank

The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only approximately 7% of proteins are observed in the corresponding PDB structures, and only approximately 25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, "Observed" (which correspond to structured regions), "Not observed" (regions with missing electron density, potentially disordered), "Uncharacterized," and "Ambiguous," depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a 'fragment' or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. "Non-observed," "Ambiguous," and "Uncharacterized" regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR(R) VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the "Observed" dataset are ordered, and that the "Not observed" regions are mostly disordered. The "Uncharacterized" regions possess some tendency toward order, whereas the predictions for the short "Ambiguous" regions are really ambiguous. Long "Ambiguous" regions (>70 amino acid residues) are mostly predicted to be ordered, suggesting that they are likely to be "wobbly" domains. Overall, we showed that completely ordered proteins are not highly abundant in PDB and many PDB sequences have disordered regions. In fact, in the analyzed dataset approximately 10% of the PDB proteins contain regions of consecutive missing or ambiguous residues longer than 30 amino-acids and approximately 40% of the proteins possess short regions (> or =10 and < 30 amino-acid long) of missing and ambiguous residues.     

Diving Into The Water: Cues Related to the Decision‐Making by an Egg Parasitoid Attacking Underwater Hosts

Although adult parasitoids spend a majority of their lives above ground, females of several species must search for their host in the water or on the soil. Adult parasitoids above ground can use a variety of sensory cues to detect their hosts from a distance. However, their sensory cues can be impaired from volatile chemicals, and their visual stimuli can be decreased while submerging or burrowing in the water of soil during their search for their hosts. Searching underwater or underground would incur high foraging costs, that is, time and energy consumption and increase risk of drowning. Therefore, to reduce such costs and increase searching efficiency, the decision on where to start submerging or burrowing for attacking hosts is important for parasitoids. Furthermore, there are no studies that have examined the cues of submerging or burrowing parasitoids on their exploit for the decision to attack their hosts. We have examined the cues used by the egg parasitoid Tiphodytes gerriphagus attacking underwater hosts. We compared the searching behaviors of T. gerriphagus among four oviposition site conditions. The four sites investigated were oviposition site with both host adult chemical residues and presence of eggs, with only the presence of eggs, with only the host adult chemical residues, and without any cue. Our results indicated that T. gerriphagus more frequently contacted and submerged at oviposition sites with the adult residues rather than at oviposition sites without them. Nonetheless, the presence of underwater host eggs did not affect the host‐searching behavior. This suggests that T. gerriphagus decided to submerge at the oviposition site in response to the adult residues. Furthermore, our observation also suggested that T. gerriphagus has already detected that the adult residues might be volatile before contacting the oviposition site. Finally, we will discuss the exploitation patterns of host‐searching cue by parasitoids that need to submerge from the context of its reliability and detectability problems.     

Three-dimensional view of the surface motif associated with the P-loop structure: cis and trans cases of convergent evolution

Here we identify the determinants of the nucleotide-binding ability associated with the P-loop-containing proteins, inferring their functional importance from their structural convergence to a unique three- dimensional (3D) motif. (1) A new surface 3D pattern is identified for the P-loop nucleotide-binding region, which is more selective than the corresponding sequence pattern; (2) the signature displays one residue that we propose is the determinant for the guanine-binding ability (the residues aligned to ras D119; this residue is known to be important only in the G-proteins, we extend the prediction to all the other P-loop- containing proteins); and (3) two cases of convergent evolution at the molecular level are highlighted in the analysis of the active site: the positive charge aligned to ras K117 and the arginine residues aligned to the GAP arginine finger.The analysis of the residues conserved on protein surfaces allows one to identify new functional or evolutionary relationships among protein structures that would not be detectable by conventional sequence or structure comparison methods.     

The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Intrinsic Disorder in the Protein Data Bank

Abstract

The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only ~7% of proteins are observed in the corresponding PDB structures, and only ~25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, “Observed” (which correspond to structured regions), “Not observed” (regions with missing electron density, potentially disordered), “Uncharacterized,” and “Ambiguous,” depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a ‘fragment’ or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. “Non-observed,” “Ambiguous,” and “Uncharacterized” regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR® VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the “Observed” dataset are ordered, and that the “Not observed” regions are mostly disordered. The “Uncharacterized” regions possess some tendency toward order, whereas the predictions for the short “Ambiguous” regions are really ambiguous. Long “Ambiguous” regions (>70 amino acid residues) are mostly predicted to be ordered, suggesting that they are likely to be “wobbly” domains.

Overall, we showed that completely ordered proteins are not highly abundant in PDB and many PDB sequences have disordered regions. In fact, in the analyzed dataset ~10% of the PDB proteins contain regions of consecutive missing or ambiguous residues longer than 30 amino-acids and ~40% of the proteins possess short regions (≥10 and <30 amino-acid long) of missing and ambiguous residues.     

Mutational alterations in the homotetrameric chaperone SecB that implicate the structure as dimer of dimers.

Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states. SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components. SecBF74I is tetrameric but binds ligand with a lower affinity. Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers. We conclude that the tetramer is a dimer of dimers and that the residues Cys76, Val78, and Gln80 must be involved either directly or indirectly in forming the interface between dimers. These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.