Biotinylation Domain Research Articles

Preferential targeting of Nav1.6 voltage-gated Na+ Channels to the axon initial segment during development.

During axonal maturation, voltage-gated sodium (Nav) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Nav reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Nav1.6 construct containing an extracellular biotinylation domain we demonstrate that Nav1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Nav1.6 channel lacking the ankyrin-binding motif nor a chimeric Kv2.1 channel containing the Nav ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Nav1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.

Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level.

Monitoring of intracellular redox status ina bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development ofa redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of Förster resonance energy transfer (FRET). For the development ofthe sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close tothe chromophore anda constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated.

Optimisation of Recombinant Production of Active Human Cardiac SERCA2a ATPase

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca2+ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.

Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli

BackgroundBiotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin.ResultsIn this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli.ConclusionsThe strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

A novel, generic and effective method for the rapid purification of G protein-coupled receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.

Solution Structure and Backbone Dynamics of the Biotinylation Domain of Helicobacter pylori Biotin-carboxyl Carrier Protein

Acetyl-CoA carboxylase (ACC) is an excellent candidate for antibiotics drug target, which mediates malonyl-CoA synthesis from acetyl-CoA through acetylation process. It is also involved in the committed step of fatty acid synthesis which is essential for living organisms. We have determined the three dimensional structure of C terminal domain of HP0371, biotin-carboxyl carrier protein of H. pyroli, in solution state using heteronuclear multi-dimensional NMR spectroscopy. The structure of HP0371 shows a flatten b-sheet fold which is similar with that of E. coli. However, the sequence and structure of protruding thumb are different with that of E. coli and the thumb shows different basis of structural rigidity based on backbone dynamics data.

Heterologous expression and comparative characterization of the human neuromedin U subtype II receptor using the methylotrophic yeast Pichia pastoris and mammalian cells

Neuromedin U (a neuropeptide) plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow and pain. The physiological actions of NmU are mediated via two recently identified G protein-coupled receptors namely the neuromedin U type 1 receptor (NmU 1R) and the neuromedin U type 2 receptor (NmU 2R). Despite their crucial roles in cell physiology, structural information on these receptors is limited, mainly due to their low expression levels in native tissues. Here, we report the overexpression of the human NmU 2R in the methylotrophic yeast Pichia pastoris and baby hamster kidney (BHK) cells using the Semliki Forest virus (SFV) system. The recombinant receptor was expressed as a fusion protein with three different affinity tags namely, the Flag tag, the histidine 10 tag and the biotinylation domain of Propionobacterium shermanii. Expression level of the recombinant receptor was 6–9 pmol/mg under optimized conditions, which is significantly higher than the expression level in the native tissues. The recombinant receptor binds to its endogenous ligand neuromedin U with high affinity (Kd = 0.8–1.0 nM) and the binding constant for the recombinant receptor is similar to that of the wild type NmU 2R. Enzymatic deglycosylation suggested that the recombinant NmU 2R was glycosylated in P. pastoris, but not in BHK cells. Confocal laser scanning microscopy and immunogold labelling experiment revealed that the recombinant receptor was predominantly localized in the intracellular membranes. To our knowledge, this is the first report of heterologous overexpression of an affinity tagged recombinant NmU 2R and it should facilitate further characterization of this receptor.

Leishmania mexicana amazonensis: Development of a peptide tag useful for labeling and purifying biotinylated recombinant proteins

A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin–horseradish peroxidase and purified on immobilized monomeric avidin resins.

Biotinylation in the hyperthermophile Aquifex aeolicus.

Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific. The exact details of the protein-protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCP Delta 67) from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCP Delta 67 at temperatures of up to 70 degrees C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus. This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCP Delta 67 complex. Furthermore, this complex was also formed between BPL and a BCCP Delta 67 mutant lacking the lysine residue (BCCP Delta 67 K117L) however, complex formation was considerably reduced using holo-BCCP Delta 67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCP Delta 67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition.

Molecular cloning and domain structure of chicken pyruvate carboxylase

Pyruvate carboxylase (PC) [EC 6.4.1.1] is a biotin-dependent carboxylase that catalyses the conversion of pyruvate to oxaloacetate. Here we have determined the complete nucleotide sequence encoding chicken PC (cPC) by screening a liver cDNA library, by RT-PCR of poly(A) + RNA, and by PCR of genomic DNA. The full-length transcript contains an open reading frame of 3537 nucleotides, including the stop codon, encoding a polypeptide of 1178 amino acids with M r of 127,262. The amino acid sequence of cPC shows approximately 77% identity to mammalian PC. Limited proteolysis of pure cPC with chymotrypsin yields a major stable 75 kDa C-terminal peptide, including the biotinyl domain and a minor, unstable 39 kDa N-terminal peptide. Northern analysis of poly(A) + RNA isolated from chicken liver has shown that cPC's mRNA is approximately 5 kb in length, including a very long 3 ′-untranslated region.

Interchangeable Enzyme Modules: FUNCTIONAL REPLACEMENT OF THE ESSENTIAL LINKER OF THE BIOTINYLATED SUBUNIT OF ACETYL-CoA CARBOXYLASE WITH A LINKER FROM THE LIPOYLATED SUBUNIT OF PYRUVATE DEHYDROGENASE

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step of fatty acid synthesis. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin. In most biotinylated proteins, the biotin moiety is attached to a lysine residue located about 35 residues from the carboxyl terminus of the protein, which lies in the center of a strongly conserved sequence that forms a tightly folded anti-parallel beta-barrel structure. Located upstream of the conserved biotinoyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers. In E. coli BCCP, this putative linker extends for about 42 residues with over half of the residues being proline or alanine. I report that deletion of the 30 linker residues located adjacent to the biotinoyl domain resulted in a BCCP species that was defective in function in vivo, although it was efficiently biotinylated. Expression of this BCCP species failed to restore normal growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperatures. In contrast, replacement of the deleted BCCP linker with a linker derived from E. coli pyruvate dehydrogenase gave a chimeric BCCP species that had normal in vivo function. Expression of BCCPs having deletions of various segments of the linker region of the chimeric protein showed that some deletions of up to 24 residues had significant or full biological activity, whereas others had very weak or no activity. The inactive deletion proteins all lacked an APAAAAA sequence located adjacent to the tightly folded biotinyl domain, whereas deletions that removed only upstream linker sequences remained active. Deletions within the linker of the wild type BCCP protein also showed that the residues adjacent to the tightly folded domain play an essential role in protein function, although in this case some proteins with deletions within this region retained activity. Retention of activity was due to fusion of the domain to upstream sequences. These data provide new evidence for the functional and structural similarities of biotinylated and lipoylated proteins and strongly support a common evolutionary origin of these enzyme subunits.

The Biotinyl Domain of Escherichia coli Acetyl-CoA Carboxylase: EVIDENCE THAT THE “THUMB” STRUCTURE IS ESSENTIAL AND THAT THE DOMAIN FUNCTIONS AS A DIMER

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the first committed step of fatty acid synthesis. Similar proteins are found in other bacteria and in chloroplasts. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin to a specific lysine residue. However, the BCCP biotinyl domain differs from many of these proteins in that an eight-amino acid residue insertion is present upstream of the biotinylated lysine. X-ray crystallographic and multidimensional NMR studies show that these residues constitute a structure that has the appearance of an extended thumb that protrudes from the otherwise highly symmetrical domain structure. I report that expression of two mutant BCCPs lacking the thumb residues fails to restore growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperature. Alignment of BCCPs from various organisms shows that only two of the eight thumb residues are strictly conserved, and amino acid substitution of either residue results in proteins giving only weak growth of the temperature-sensitive E. coli strain. Therefore, the thumb structure is essential for the function of BCCP in the ACC reaction and provides a useful motif for distinguishing the biotinylated proteins of multisubunit ACCs from those of enzymes catalyzing other biotin-dependent reactions. An unexpected result was that expression of a mutant BCCP in which the biotinylated lysine residue was substituted with cysteine was able to partially restore growth and fatty acid synthesis to the temperature-sensitive E. coli strain. This complementation was shown to be specific to BCCPs having native structure (excepting the biotinylated lysine) and is interpreted in terms of dimerization of the BCCP biotinyl domain during the ACC reaction.

External Surface Display of Proteins Linked to DNA-Binding Domains

A novel system (DBDX) was developed which allows the external surface display on filamentous bacteriophage of proteins fused to either the N- or the C-terminus of a DNA-binding protein. In conjunction with helper phage infection, expression of proteins fused to the estrogen receptor DNA-binding domain (DBD) in a phagemid vector containing the DNA sequence recognized by the DBD resulted in the production of phage particles which display the fusion protein through the phage pVIII coat on the external surface of the particle. The viability of the technique was established with several model systems: particles displaying the C-terminal domain of N-cadherin or the biotinylation domain of propionyl coenzyme A carboxylase fused to the C-terminus of the DBD were found to be bound specifically by antibody or streptavidin, respectively. Human κ constant region cDNA was selected from a N-terminal DBD fusion lymphocyte cDNA library after two rounds of selection with anti-κ antibody. This display system may complement currently available bacterial selection techniques.

Expression in Escherichia coli of N- and C-terminally Deleted Human Holocarboxylase Synthetase: INFLUENCE OF THE N-TERMINUS ON BIOTINYLATION AND IDENTIFICATION OF A MINIMUM FUNCTIONAL PROTEIN

Biotin functions as a covalently bound cofactor of biotindependent carboxylases. Biotin attachment is catalyzed by biotin protein ligases, called holocarboxylase synthetase in mammals and BirA in prokaryotes. These enzymes show a high degree of sequence similarity in their biotinylation domains but differ markedly in the length and sequence of their N terminus. BirA is also the repressor of the biotin operon, and its DNA attachment site is located in its N terminus. The function of the eukaryotic N terminus is unknown. Holocarboxylase synthetase with N- and C-terminal deletions were evaluated for the ability to catalyze biotinylation after expression in Escherichia coli using bacterial and human acceptor substrates. We showed that the minimum functional protein is comprised of the last 349 of the 726-residue protein, which includes the biotinylation domain. Significantly, enzyme containing intermediate length, N-terminal deletions interfered with biotin transfer and interaction with different peptide acceptor substrates. We propose that the N terminus of holocarboxylase synthetase contributes to biotinylation through N- and C-terminal interactions and may affect acceptor substrate recognition. Our findings provide a rationale for the biotin responsiveness of patients with point mutations in the N-terminal sequence of holocarboxylase synthetase. Such mutant enzyme may respond to biotin-mediated stabilization of the substrate-bound complex.

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for post-translational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight β-turn that houses the lysine residue targetted for biotinylation, and the β-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct post-translational modification.

Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability

Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S. cerevisiae permits a one-step purification via a biotinylation domain. Milligram amounts of the protein are obtained starting from 2 l of yeast culture. The HUP1 protein is used as a model eukaryotic membrane protein of the ‘major facilitator superfamily’ (MFS) to study specific lipid requirements for activity and stability. Testing two series of detergents revealed that n-nonyl-β- D-glucoside (NG) and n-octyl-β- D-glucoside (OG) solubilize the HUP1 protein efficiently. Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids. When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected. These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG. Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein. The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family.

Structure and selectivity in post-translational modification: attaching the biotinyl–lysine and lipoyl–lysine swinging arms in multifunctional enzymes

The post-translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding beta-turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed by two different ligases. We have expressed in Escherichia coli a sub-gene encoding the biotinyl domain of E.coli acetyl-CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro. The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process. LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain beta-turn, but was responsive to structural cues in the flanking beta-strands. BPL was much less sensitive to changes in these beta-strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain beta-turn. The presence of a further protruding thumb between the beta2 and beta3 strands in the wild-type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli. The structural basis of this discrimination contrasts with other forms of post-translational modification, where the sequence motif surrounding the target residue can be the principal determinant.

Coding Sequence Mutations in the Alpha Subunit of Propionyl-CoA Carboxylase in Patients with Propionic Acidemia

Propionic acidemia is a rare autosomal recessive disorder of intermediary metabolism. It is caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric protein composed of two subunits, α and β. PCC requires ATP and biotin as cofactors for the reaction, the latter enzymatically added onto the α subunit. We investigated coding sequence mutations in the α subunit of PCC by analyzing fibroblast RNA from propionic acidemia patients deficient in α subunit function by single-strand conformation polymorphism and direct sequencing. Five missense mutations and one short in-frame deletion were found among different patients. Four mutations were located in the putative biotin carboxylase domain, whereas the two others were within the 67-amino-acid C-terminal domain previously shown to be required to obtain biotinylation of the α subunit. We analyzed fibroblast extracts for the presence of a biotinylated α subunit by Western blot analysis using streptavidin coupled to alkaline phosphatase. Four of five cell lines failed to show a biotinylated α subunit, regardless of the position of the mutations within the coding sequence. Two mutations located in the biotinylation domain were expressed in anEscherichia coli-based system and shown to abolish biotinylation of the domain. The results suggest that most mutations have a severe impact on the stability or the functionality of the α subunit.

Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation

Phage display is a powerful technique for identifying specific ligands to a given target. In this work random peptides derived from the biotin accepting domain of the Klebsiella pneumoniae oxaloacetate decarboxylase were displayed on bacteriophage lambda heads to determine the minimal sequence length that is necessary to effect biotinylation in vivo. Phages with a functional biotinylation domain were identified after affinity purification with immobilised avidin. All biotinylated phages isolated this way were found to have a sequence of 66 amino acids from the parental protein in common. This minimal biotinylation domain is fully functional as a biotin acceptor and more resistant to proteolytic attack compared to domains of larger size derived from the same protein. The data present the first example of a posttranslational protein modification analysed in a phage display system. Moreover, a biotin domain of reduced size and improved stability was identified, that should be superior to the larger parental protein as a tag to generate biotinylated fusion proteins.

Selectivity of post-translational modification in biotinylated proteins: the carboxy carrier protein of the acetyl-CoA carboxylase of Escherichia coli.

Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two beta-sheets that make up the domain. A sub-gene encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E. coli. The biotinyl domain was readily released by cleavage with thrombin. Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes. No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro. The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated. It was also a poor substrate for lipoylation in vitro catalysed by the E. coli lipoyl protein ligase encoded by the lplA gene. The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes. The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E. coli but probably by a pathway different from that mediated by the lplA-dependent ligase. The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains.