David K. Wilson

The Structure of Aip1p, a WD Repeat Protein That Regulates Cofilin-mediated Actin Depolymerization

Actin-interacting protein 1 (Aip1p) is a 67-kDa WD repeat protein known to regulate the depolymerization of actin filaments by cofilin and is conserved in organisms ranging from yeast to mammals. The crystal structure of Aip1p from Saccharomyces cerevisiae was determined to a 2.3-Å resolution and a final crystallographic R-factor of 0.204. The structure reveals that the overall fold is formed by two connected seven-bladed β-propellers and has important implications for the structure of Aip1 from other organisms and WD repeat-containing proteins in general. These results were unexpected because a maximum of 10 WD repeats had been reported in the literature for this protein using sequence data. The surfaces of the β-propellers formed by the D-A and B-C loops are positioned adjacent to one another, giving Aip1p a shape that resembles an open “clamshell.” The mapping of conserved residues to the structure of Aip1p reveals dense patches of conserved residues on the surface of one β-propeller and at the interface of the two β-propellers. These two patches of conserved residues suggest a potential binding site for F-actin on Aip1p and that the orientation of the β-propellers with respect to one another plays a role in binding an actin-cofilin complex. In addition, the conserved interface between the domains is mediated by a number of interactions that appear to impart rigidity between the two domains of Aip1p and may make a large substrate-induced conformational change difficult.

Specifically Targeted Modification of Human Aldose Reductase by Physiological Disulfides

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.

The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure

Ski8p is a 44‐kD protein that primarily functions in the regulation of exosome‐mediated, 3′→ 5′ degradation of damaged mRNA. It does so by forming a complex with two partner proteins, Ski2p and Ski3p, which complete a complex that is capable of recruiting and activating the exosome/Ski7p complex that functions in RNA degradation. Ski8p also functions in meiotic recombination in complex with Spo11 in yeast. It is one of the many hundreds of primarily eukaryotic proteins containing tandem copies of WD repeats (also known as WD40 or β‐transducin repeats), which are short ∼40 amino acid motifs, often terminating in a Trp–Asp dipeptide. Genomic analyses have demonstrated that WD repeats are found in 1%–2% of proteins in a typical eukaryote, but are extremely rare in prokaryotes. Almost all structurally characterized WD‐repeat proteins are composed of seven such repeats and fold into seven‐bladed β propellers. Ski8p was thought to contain five WD repeats on the basis of primary sequence analysis implying a five‐bladed propeller. The 1.9 Å crystal structure unexpectedly exhibits a seven‐bladed propeller fold with seven structurally authentic WD repeats. Structure‐based sequence alignments show additional sequence diversity in the two undetected repeats. This demonstrates that many WD repeats have not yet been identified in sequences and also raises the possibility that the seven‐bladed propeller may be the predominant fold for this family of proteins.

Refined 2.5Åstructure of murine adenosine deaminase at pH 6.0

The X-ray structure of murine adenosine deaminase complexed with the transition-state analogue 6-hydroxyl-1,6-dihydropurine ribonucleoside has been determined from a single crystal grown at pH 4.2 and transferred to mother liquor of increasing pH up to a final pH of 6.0 prior to data collection. The structure has been refined to 2.5 A to a final crystallographic R-factor of 20% using phases from the previously refined 2.4 A structure at pH 4.2. Kinetic measurements show that the enzyme is only 20% active at pH 4.2 whereas it is fully active between pH 6.0 and pH 8.5. The refined structures at either pH are essentially the same. Consideration of the pKa values of the key catalytic residues and the mechanism proposed on the basis of the structure suggests that the ionization state of these residues is largely responsible for the pH dependence on activity.

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+

Aldo‐keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X‐ray crystal structures of Lys‐274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP+‐bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water‐mediated interactions between Glu‐227, Lys‐274 and the cofactor 2′‐phosphate and 3′‐hydroxy groups. Because anchoring contacts of its Glu‐227 are lost, the coenzyme‐enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild‐type remains partly disordered in the NADP+‐bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild‐type.

Catalytic mechanism and substrate selectivity of aldo‐keto reductases: Insights from structure‐function studies of Candida tenuis xylose reductase

Aldo‐keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)‐dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure‐function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the ε‐NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular‐level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications. iubmb Life, 58: 499‐507, 2006

Identification of Formyl Kynurenine Formamidase and Kynurenine Aminotransferase from Saccharomyces cerevisiae Using Crystallographic, Bioinformatic and Biochemical Evidence

The essential enzymatic cofactor NAD+ can be synthesized in many eukaryotes, including Saccharomyces cerevisiae and mammals, using tryptophan as a starting material. Metabolites along the pathway or on branches have important biological functions. For example, kynurenic acid can act as an NMDA antagonist, thereby functioning as a neuroprotectant in a wide range of pathological states. N-Formyl kynurenine formamidase (FKF) catalyzes the second step of the NAD+ biosynthetic pathway by hydrolyzing N-formyl kynurenine to produce kynurenine and formate. The S. cerevisiae FKF had been reported to be a pyridoxal phosphate-dependent enzyme encoded by BNA3. We used combined crystallographic, bioinformatic and biochemical methods to demonstrate that Bna3p is not an FKF but rather is most likely the yeast kynurenine aminotransferase, which converts kynurenine to kynurenic acid. Additionally, we identify YDR428C, a yeast ORF coding for an alpha/beta hydrolase with no previously assigned function, as the FKF. We predicted its function based on our interpretation of prior structural genomics results and on its sequence homology to known FKFs. Biochemical, bioinformatics, genetic and in vivo metabolite data derived from LC-MS demonstrate that YDR428C, which we have designated BNA7, is the yeast FKF.

Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis

Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/K(m)) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 A (1 A=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8-9 kJ/mol.

The 1.1-Å Structure of the Spindle Checkpoint Protein Bub3p Reveals Functional Regions

Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and β-transducin repeats), short sequence motifs appearing in clusters of 4–16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 Å and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure.

Pseudomonas fluorescens mannitol 2-dehydrogenase and the family of polyol-specific long-chain dehydrogenases/reductases: sequence-based classification and analysis of structure-function relationships.

Multiple sequence alignment and analysis of evolutionary relationships have been used to characterize a family of polyol-specific long-chain dehydrogenases/reductases (PSLDRs). At the present time, 66 known and putative NAD(P)H-dependent oxidoreductases of mainly prokaryotic origin and between 357 and 544 amino acids in length constitute this family. The family is shown to include D-mannitol 2-dehydrogenase, D-mannonate 5-oxidoreductase, D-altronate 5-oxidoreductase, D-arabinitol 4-dehydrogenase, and D-mannitol-1-phosphate 5-dehydrogenase which form individual sub-families (defined by internal sequence identity of >/=30%) having distant origin and divergent substrate specificity but clearly displaying entire-chain relationship. When all forms are aligned, only three residues, Gly-33, Asp-230, and Lys-295 (in the numbering of Pseudomonas fluorescens D-mannitol 2-dehydrogenase (PsM2DH)) are strictly conserved. By combining sequence alignment with the known structure of PsM2DH and results from site-directed mutagenesis, we have developed a structure/function analysis for the family. Gly-33 is in the N-terminal coenzyme-binding domain and part of a nucleotide fingerprint region for the family, and Asp-230 and Lys-295 are at an interdomain segment contributing to the active site in which the lysine likely functions as the catalytic general acid/base. PSLDRs do not require a metal cofactor for activity and are specific for transferring the 4-pro-S hydrogen from NAD(P)H. Comparisons reveal that the core part of the two-domain fold has been conserved throughout all family members, perhaps reflecting the recruitment of a stable oxidoreductase structure and extensive trimming thereof to acquire functional properties specific to each sub-family. They also identify interactions that define the chemical mechanism of oxidoreduction and likely contribute to substrate and co-substrate specificities and are thus relevant for protein engineering.