Biodegradation of all stereoisomers of the EDTA substitute iminodisuccinate by Agrobacterium tumefaciens BY6 requires an epimerase and a stereoselective C-N lyase

Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe(2+)-, Mg(2+)-, and Ca(2+)-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn(2+)-IDS were rather scant and very slow on Cu(2+)-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating d-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase.     

Sequencing and heterologous expression of an epimerase and two lyases from iminodisuccinate-degrading bacteria

Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl(B) and icl(S), encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl(B) gene was chromosomal like the corresponding icl(S) gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.     

The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: insights into its biological function

In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.     

Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5'-phosphate-dependent enzymes.

The three-dimensional structure of diaminopelargonic acid synthase, a vitamin B6-dependent enzyme in the pathway of the biosynthesis of biotin, has been determined to 1.8 A resolution by X-ray crystallography. The structure was solved by multi-wavelength anomalous diffraction techniques using a crystal derivatized with mercury ions. The protein model has been refined to a crystallographic R -value of 17.5% (R -free 22.6%). Each enzyme subunit consists of two domains, a large domain (residues 50-329) containing a seven-stranded predominantly parallel beta-sheet, surrounded by alpha-helices, and a small domain comprising residues 1-49 and 330-429. Two subunits, related by a non-crystallographic dyad in the crystals, form the homodimeric molecule, which contains two equal active sites. Pyridoxal-5'-phosphate is bound in a cleft formed by both domains of one subunit and the large domain of the second subunit. The cofactor is anchored to the enzyme by a covalent linkage to the side-chain of the invariant residue Lys274. The phosphate group interacts with main-chain nitrogen atoms and the side-chain of Ser113, located at the N terminus of an alpha-helix. The pyridine nitrogen forms a hydrogen bond to the side-chain of the invariant residue Asp245. Electron density corresponding to a metal ion, most likely Na(+), was found in a tight turn at the surface of the enzyme. Structure analysis reveals that diaminopelargonic acid synthase belongs to the family of vitamin B6-dependent aminotransferases with the same fold as originally observed in aspartate aminotransferase. A multiple structure alignment of enzymes in this family indicated that they form at least six different subclasses. Striking differences in the fold of the N-terminal part of the polypeptide chain are one of the hallmarks of these subclasses. Diaminopelargonic acid synthase is a member of the aminotransferase subclass III. From the structure of the non-productive complex of the holoenzyme with the substrate 7-keto-8-aminopelargonic acid the location of the active site and residues involved in substrate binding have been identified.     

Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production

Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 A resolution. The subunit structure of ECAI is organised into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.     

Structure of the ribosome associating GTPase HflX

The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline

The crystal structure of GcnA, an N-acetyl-β-d-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal α-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (β/α)₈ TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-β-d-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.     

Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase

In chlorophyll biosynthesis, insertion of Mg(2+) into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A resolution to the crystallographic R-factor of 22.2 % (R(free)=24.5 %). It belongs to the chaperone-like "ATPase associated with a variety of cellular activities" (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase.     

The crystal structure of leucyl/phenylalanyl-tRNA-protein transferase from Escherichia coli

Leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is an N-end rule pathway enzyme, which catalyzes the transfer of Leu and Phe from aminoacyl-tRNAs to exposed N-terminal Arg or Lys residues of acceptor proteins. Here, we report the 1.6 A resolution crystal structure of L/F-transferase (JW0868) from Escherichia coli, the first three-dimensional structure of an L/F-transferase. The L/F-transferase adopts a monomeric structure consisting of two domains that form a bilobate molecule. The N-terminal domain forms a small lobe with a novel fold. The large C-terminal domain has a highly conserved fold, which is observed in the GCN5-related N-acetyltransferase (GNAT) family. Most of the conserved residues of L/F-transferase reside in the central cavity, which exists at the interface between the N-terminal and C-terminal domains. A comparison of the structures of L/F-transferase and the bacterial peptidoglycan synthase FemX, indicated a structural homology in the C-terminal domain, and a similar domain interface region. Although the peptidyltransferase function is shared between the two proteins, the enzymatic mechanism would differ. The conserved residues in the central cavity of L/F-transferase suggest that this region is important for the enzyme catalysis.     

Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae--the prototype of the DEAD box protein family.

BACKGROUND: Translation initiation factor 4A (elF4A) is the prototype of the DEAD-box family of proteins. DEAD-box proteins are involved in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. Energy from ATP hydrolysis is used to perform RNA unwinding during initiation of mRNA translation. The presence of elF4A is required for the 43S preinitiation complex to bind to and scan the mRNA. RESULTS: We present here the crystal structure of the nucleotide-binding domain of elF4A at 2.0 A and the structures with bound adenosinediphosphate and adenosinetriphosphate at 2.2 A and 2.4 A resolution, respectively. The structure of the apo form of the enzyme has been determined by multiple isomorphous replacement. The ATPase domain contains a central seven-stranded beta sheet flanked by nine alpha helices. Despite low sequence homology to the NTPase domains of RNA and DNA helicases, the three-dimensional fold of elF4A is nearly identical to the DNA helicase PcrA of Bacillus stearothermophilus and to the RNA helicase NS3 of hepatitis C virus. CONCLUSIONS: We have determined the crystal structure of the N-terminal domain of the elF4A from yeast as the first structure of a member of the DEAD-box protein family. The complex of the protein with bound ADP and ATP offers insight into the mechanism of ATP hydrolysis and the transfer of energy to unwind RNA. The identical fold of the ATPase domain of the DNA helicase PcrA of B. stearothermophilus and the RNA helicase of hepatitis C virus suggests a common fold for all ATPase domains of DExx- and DEAD-box proteins.     

X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil

Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.     

A novel fold revealed by Mycobacterium tuberculosis NAD kinase, a key allosteric enzyme in NADP biosynthesis

NAD kinase catalyzes the magnesium-dependent phosphorylation of NAD, representing the sole source of freshly synthesized NADP in all organisms. The enzyme is essential for the growth of the deadly multidrug-resistant pathogen Mycobacterium tuberculosis and is an attractive target for novel antitubercular agents. The crystal structure of NAD kinase has been solved by multiwavelength anomalous dispersion at a resolution of 2.3 A in its T state. Two crystal forms have been obtained revealing either a dimer or a tetramer. The enzyme architecture discloses a novel molecular arrangement, with each subunit consisting of an alpha/beta N-terminal domain and a C-terminal 12-stranded beta sandwich domain, connected by swapped beta strands. The C-terminal domain shows a striking internal approximate 222 symmetry and an unprecedented topology, revealing a novel fold within the family of all beta structures. The catalytic site is located in the long crevice that defines the interface between the domains. The conserved GGDG structural fingerprint of the catalytic site is reminiscent of the related region in 6-phosphofructokinase, supporting the hypothesis that NAD kinase belongs to a newly reported superfamily of kinases.     

Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit

The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.     

Crystal structure of YHI9, the yeast member of the phenazine biosynthesis PhzF enzyme superfamily

In the Pseudomonas bacterial genomes, the PhzF proteins are involved in the production of phenazine derivative antibiotic and antifungal compounds. The PhzF superfamily however also encompasses proteins in all genomes from bacteria to eukaryotes, for which no function has been assigned. We have determined the three dimensional crystal structure at 2.05 A resolution of YHI9, the yeast member of the PhzF family. YHI9 has a fold similar to bacterial diaminopimelate epimerase, revealing a bimodular structure with an internal symmetry. Residue conservation identifies a putative active site at the interface between the two domains. Evolution of this protein by gene duplication, gene fusion and domain swapping from an ancestral gene containing the "hot dog" fold, identifies the protein as a "kinked double hot dog" fold.     

Crystal Structure of Diaminopimelate Epimerase from Arabidopsis thaliana, an Amino Acid Racemase Critical for l-Lysine Biosynthesis

Diaminopimelate (DAP) epimerase is a key enzyme for the biosynthesis of lysine in plants. Lysine is an essential dietary nutrient for mammals. In both plants and bacteria, DAP epimerase catalyzes the interconversion of ll-DAP and dl(meso)-DAP. The absence of a mammalian homolog makes DAP epimerase a promising target for the design of novel herbicides and antibacterials. This enzyme requires no cofactors and it functions through an unusual mechanism involving two cysteine residues acting in concert and alternating as a base (thiolate) and as an acid (thiol). The present study reports the crystal structures of two enzyme-inhibitor complexes of DAP epimerase from Arabidopsis thaliana with different isomers of the irreversible inhibitor and substrate mimic, 2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate, at 1.95 and 2.3 Å resolution. These structures provide the first atomic details of a plant amino acid racemase. Structural analysis reveals that ligand binding to a cleft between the two domains of the enzyme is accompanied by domain closure with two strictly conserved cysteine residues, Cys99 and Cys254, optimally positioned to perform acid/base catalysis via a carbanion stabilization mechanism on the stereogenic α-carbon atom of the amino acid. Stereochemical control in catalysis is achieved by means of a highly symmetric catalytic site that can accommodate both the l and d stereogenic centers of DAP at the proximal site, whereas specific interactions at the distal site require only the l configuration. Structural comparisons of the plant enzyme with its bacterial counterpart from Haemophilus influenzae reveal significant conservation of amino acid residues around the active site that extends to their three-dimensional structures and catalytic mechanism.     

A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase

Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 A and 2.5 A resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic alpha(3)beta(8)alpha(4) sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the beta sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta(5)beta(4) sandwich fold and three alpha helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P. The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres, which is possibly involved in the cooperativity for glutamate.     

Crystal structures of the PLP- and PMP-bound forms of BtrR, a dual functional aminotransferase involved in butirosin biosynthesis

The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)-dependent transamination reaction both of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and of amino-dideoxy-scyllo-inosose to 2-DOS. The high-resolution crystal structures of the PLP- and PMP-bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 A and 1.7 A with R(factor)/R(free) values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an alpha helical N-terminus, a central alphabetaalpha sandwich domain and an alphabeta C-terminal domain. The structures of the PLP- and PMP-bound enzymes are very similar; however BtrR-PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side-chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three-dimensional structure of an enzyme from the butirosin biosynthesis gene cluster.     

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.