J. Mitchell Guss

Structure of oxidized poplar plastocyanin at 1·6 Å resolution

The structure of poplar plastocyanin in the oxidized (CuII) state at pH 6.0 has been refined, using 1.6 A resolution counter data. The starting co-ordinates were obtained from the 2.7 A electron density map computed with phases derived by the multiple isomorphous replacement method. The model was refined successively by constrained real space, unrestrained reciprocal space, and restrained reciprocal space least-squares methods. The final residual R value is 0.17 for 8285 reflections (I greater than 2 sigma (I)). It is estimated that the root-mean-square standard deviation of the atomic positions is 0.1 A when averaged over all atoms, and 0.05 A for the Cu ligand atoms alone. The refined structure retains all the essential features of the 2.7 A model. The co-ordination geometry of the copper atom is confirmed as being distorted tetrahedral. The two Cu-N(His) bonds, 2.10 and 2.04 A, are within the range normally found in low molecular weight CuII complexes with Cu-N(imidazole) bonds. The Cu-S(Cys) bond, 2.13 A, is also normal, but the Cu-S(Met) bond, 2.90 A, is sufficiently long to raise important questions about its significance. The hydrogen-bonding and secondary structure can now be assigned confidently. Forty-four water molecules are included in the final model. Repetition of the refinement, using new data to 1.9 A resolution recorded from crystals at pH 4.2, has led to a residual R value of 0.16 for 6060 reflections (I greater than sigma (I)). There are few significant changes in the structure of poplar CuII-plastocyanin between pH 6.0 and pH 4.2. In particular, the geometry of the copper site is not affected. The observed changes in redox behaviour of plastocyanin at low pH are therefore unlikely to be connected with structural changes in the oxidized form of the protein. A number of features of the molecular structure appear to be directly related to the function of plastocyanin as an electron carrier in photosynthesis. Comparison between the known amino acid sequences of 67 plant plastocyanins reveals 52 conserved and 11 conservatively substituted residues in a total of 99. If three algal plastocyanin sequences are included in the comparison, there are still 26 conserved and 12 conservatively substituted residues. In many cases, the importance of these residues in determining the tertiary structure can be rationalized.

Structure of a human lysosomal sulfatase

BACKGROUND . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex.

Nuclear LIM‐only (LMO) and LIM‐homeodomain (LIM‐HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain‐binding protein‐1 (Ldb1). We have determined a high‐resolution X‐ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1‐LID, providing the first example of a tandem LIM:Ldb1‐LID complex and the first structure of a type‐B LIM domain. The complex possesses a highly modular structure with Ldb1‐LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone–backbone hydrogen bonds. A mutagenic screen of Ldb1‐LID, assessed by yeast two‐hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM‐HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.

Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

LIM‐homeodomain (LIM‐HD) transcription factors form a combinatorial ‘LIM code’ that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain‐binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet‐1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30‐residue Lhx3‐binding domain on Isl1 (Isl1LBD). Although the LIM interaction domain of Ldb1 (Ldb1LID) and Isl1LBD share low levels of sequence homology, X‐ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1LBD mimics Ldb1LID. These data provide a structural basis for the formation of cell type‐specific protein–protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5' splice site-like sequences.

The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(Nx)AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5′ splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine “ladder.” A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH.

Background: Bacteria need iron from the host to establish infection. Results: We report the first structure of hemoglobin bound to a bacterial protein and show that targeted disruption of this interaction can reduce Staphylococcus aureus growth when hemoglobin is the sole iron source. Conclusion: Physical capture of hemoglobin is important for iron uptake by S. aureus. Significance: Hemoglobin receptors may be targets for new antibacterial agents. Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

Potassium-activated GTPase Reaction in the G Protein-coupled Ferrous Iron Transporter B

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe2+). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe2+ membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2′-(or -3′)-O-(N-methylanthraniloyl)-β,γ-imidoguanosine 5′-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K+, which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K+ coordination, and mutation of this residue abolished K+-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K+-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.

Stabilization of a binary protein complex by intein-mediated cyclization

The study of protein–protein interactions can be hampered by the instability of one or more of the protein complex components. In this study, we showed that intein‐mediated cyclization can be used to engineer an artificial intramolecular cyclic protein complex between two interacting proteins: the largely unstable LIM‐only protein 4 (LMO4) and an unstructured domain of LIM domain binding protein 1 (ldb1). The X‐ray structure of the cyclic complex is identical to noncyclized versions of the complex. Chemical and thermal denaturation assays using intrinsic tryptophan fluorescence and dynamic light scattering were used to compare the relative stabilities of the cyclized complex, the intermolecular (or free) complex, and two linear versions of the intramolecular complex (in which the interacting domains of LMO4 and ldb1 were fused, via a flexible linker, in either orientation). In terms of resistance to denaturation, the cyclic complex is the most stable variant and the intermolecular complex is the least stable; however, the two linear intramolecular variants show significant differences in stability. These differences appear to be related to the relative contact order (the average distance in sequence between residues that make contacts within a structure) of key binding residues at the interface of the two proteins. Thus, the restriction of the more stable component of a complex may enhance stability to a greater extent than restraining less stable components.

Structural Basis for Partial Redundancy in a Class of Transcription Factors, the LIM Homeodomain Proteins, in Neural Cell Type Specification

Background: Lhx and Isl proteins contribute to genetic control in developing neurons. Results: The Lhx3/4-binding motif in Isl2 was identified, and the structures of Lhx-Isl complexes were characterized and compared. Conclusion: There are minor differences in the structures of Lhx3/4 binding Isl1/2 reflected by mutational and biophysical analyses. Significance: Redundant sets of interactions conserve function in developing neurons while allowing divergence in other contexts. Combinations of LIM homeodomain proteins form a transcriptional “LIM code” to direct the specification of neural cell types. Two paralogous pairs of LIM homeodomain proteins, LIM homeobox protein 3/4 (Lhx3/Lhx4) and Islet-1/2 (Isl1/Isl2), are expressed in developing ventral motor neurons. Lhx3 and Isl1 interact within a well characterized transcriptional complex that triggers motor neuron development, but it was not known whether Lhx4 and Isl2 could participate in equivalent complexes. We have identified an Lhx3-binding domain (LBD) in Isl2 based on sequence homology with the Isl1LBD and show that both Isl2LBD and Isl1LBD can bind each of Lhx3 and Lhx4. X-ray crystal- and small-angle x-ray scattering-derived solution structures of an Lhx4·Isl2 complex exhibit many similarities with that of Lhx3·Isl1; however, structural differences supported by mutagenic studies reveal differences in the mechanisms of binding. Differences in binding have implications for the mode of exchange of protein partners in transcriptional complexes and indicate a divergence in functions of Lhx3/4 and Isl1/2. The formation of weaker Lhx·Isl complexes would likely be masked by the availability of the other Lhx·Isl complexes in postmitotic motor neurons.