Structure and function of eukaryotic NAD(P)H:nitrate reductase

Pyridine nucleotide-dependent nitrate reductases (NRs; EC 1.6.6.1–3) are molybdenum-containing enzymes found in eukaryotic organisms which assimilate nitrate. NR is a homodimer with an ∼100 kDa polypeptide which folds into stable domains housing each of the enzyme's redox cofactors—FAD, heme-Fe molybdopterin (Mo-MPT) and the electron donor NAD(P)H—and there is also a domain for the dimer interface. NR has two active sites: the nitrate-reducing Mo-containing active site and the pyridine nucleotide active site formed between the FAD and NAD(P)H domains. The major barriers to defining the mechanism of catalysis for NR are obtaining the detailed three-dimensional structures for oxidized and reduced enzyme and more in-depth analysis of electron transfer rates in holo-NR. Recombinant expression of holo-NR and its fragments, including site-directed mutagenesis of key acative site and domain interface residues, are expected to make large contributions to this effort to understand the catalytic mechanism of NR.     

Multiple catalytically active thioredoxin folds: a winning strategy for many functions

The Thioredoxin (Trx) fold is a versatile protein scaffold consisting of a four-stranded β-sheet surrounded by three α-helices. Various insertions are possible on this structural theme originating different proteins, which show a variety of functions and specificities. During evolution, the assembly of different Trx fold domains has been used many times to build new multi-domain proteins able to perform a large number of catalytic functions. To clarify the interaction mode of the different Trx domains within a multi-domain structure and how their combination can affect catalytic performances, in this review, we report on a structural and functional analysis of the most representative proteins containing more than one catalytically active Trx domain: the eukaryotic protein disulfide isomerases (PDIs), the thermophilic protein disulfide oxidoreductases (PDOs) and the hybrid peroxiredoxins (Prxs).     

KIF5C: A new binding partner for protein kinase CK2 with a preference for the CK2α’ subunit

Protein kinase CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in eukaryotic cells. CK2 is a constitutively active tetrameric enzyme composed of two catalytic α and/or α’-subunits and two regulatory β-subunits. There is increasing evidence that the individual subunits may have independent functions and that they are asymmetrically distributed inside the cell. To gain a better understanding of the functions of the individual subunits, we employed a yeast-two-hybrid screen with CK2α and CK2α’. We identified the motor neuron protein KIF5C as a new binding partner for CK2. The interaction found in the yeast-two-hybrid screen was confirmed by co-sedimentation analysis on a sucrose density gradient and by co-immunoprecipitation analysis. Pull-down experiments and surface plasmon resonance spectrometry revealed a direct binding of KIF5C to CK2α’. Co-localization studies with neuroblastoma cells, bone marrow and with primary neurons confirmed the biochemical analysis that KIF5C preferentially bound to CK2α’. Received 8 August 2008; received after revision 3 November 2008; accepted 4 November 2008     

Ligand recognition by the I domain-containing integrins

Seven of the integrin α subunits described to date, α ₁ , α ₂ , α _L , α _X , α _d , α _M and α _E , contain a highly conserved I (or A) domain of approximately 200 amino acid residues inserted near the amino-terminus of the subunit. As the result of a variety of independent experimental approaches, a large body of data has recently accumulated that indicates that the I domains are independent, autonomously folding domains capable of directly binding ligands that play a necessary and important role in ligand binding by the intact integrins. Recent crystallographic studies have elucidated the structures of recombinant α _M and α _L I domains and also delineated a novel divalent cation-binding motif within the I domains (metal ion-dependent adhesion site, MIDAS) that appears to mediate the divalent cation binding of the I domains and the I domain-containing integrins to their ligands.     

SET domain protein lysine methyltransferases: Structure, specificity and catalysis

Site- and state-specific lysine methylation of histones is catalyzed by a family of proteins that contain the evolutionarily conserved SET domain and plays a fundamental role in epigenetic regulation of gene activation and silencing in all eukaryotes. The recently determined three-dimensional structures of the SET domains from chromosomal proteins reveal that the core SET domain structure contains a two-domain architecture, consisting of a conserved anti-parallel β-barrel and a structurally variable insert that surround a unusual knot-like structure that comprises the enzyme active site. These structures of the SET domains, either in the free state or when bound to cofactor S-adenosyl-L-homocysteine and/or histone peptide, mimicking an enzyme/cofactor/substrate complex, further yield the structural insights into the molecular basis of the substrate specificity, methylation multiplicity and the catalytic mechanism of histone lysine methylation. Received 10 June 2006; accepted 22 August 2006     

The prolyl oligopeptidase family

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze peptides containing more than about 30 residues. This group is unrelated to the classical trypsin and subtilisin families, and includes dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B, in addition to the prototype prolyl oligopeptidase. The recent crystal structure determination of prolyl oligopeptidase (80 kDa) has shown that the enzyme contains a peptidase domain with an α/β hydrolase fold, and its catalytic triad is covered by the central tunnel of an unusual seven-bladed β-propeller. This domain operates as a gating filter, excluding large, structured peptides from the active site. The binding mode of substrates and the catalytic mechanism differ from that of the classical serine peptidases in several features. The members of the family are important targets of drug design. Prolyl oligopeptidase is involved in amnesia, depression and blood pressure control, dipeptidyl peptidase IV in type 2 diabetes and oligopeptidase B in trypanosomiasis. Received 8 August 2001; received after revision 19 September 2001; accepted 21 September 2001     

The structural intolerance of the PrP α-fold for polar substitution of the helix-3 methionines

The conversion of the cellular prion protein (PrP^C) into its disease-associated form (PrP^Sc) involves a major conformational change and the accumulation of sulfoxidized methionines. Computational and synthetic approaches have shown that this change in the polarity of M206 and M213 impacts the C-terminal domain native α-fold allowing the flexibility required for the structural conversion. To test the effect in the full-length molecule with site-specificity, we have generated M-to-S mutations. Molecular dynamics simulations show that the replacement indeed perturbs the native state. When this mutation is placed at the conserved methionines of HaPrP(23–231), only substitutions at the Helix-3 impair the α-fold, stabilizing a non-native state with perturbed secondary structure, loss of native tertiary contacts, increased surface hydrophobicity, reduced thermal stability and an enhanced tendency to aggregate into protofibrillar polymers. Our work supports that M206 and M213 function as α-fold gatekeepers and suggests that their redox state regulate misfolding routes.     

Fractal symmetry of protein interior: what have we learned?

The application of fractal dimension-based constructs to probe the protein interior dates back to the development of the concept of fractal dimension itself. Numerous approaches have been tried and tested over a course of (almost) 30 years with the aim of elucidating the various facets of symmetry of self-similarity prevalent in the protein interior. In the last 5 years especially, there has been a startling upsurge of research that innovatively stretches the limits of fractal-based studies to present an array of unexpected results on the biophysical properties of protein interior. In this article, we introduce readers to the fundamentals of fractals, reviewing the commonality (and the lack of it) between these approaches before exploring the patterns in the results that they produced. Clustering the approaches in major schools of protein self-similarity studies, we describe the evolution of fractal dimension-based methodologies. The genealogy of approaches (and results) presented here portrays a clear picture of the contemporary state of fractal-based studies in the context of the protein interior. To underline the utility of fractal dimension-based measures further, we have performed a correlation dimension analysis on all of the available non-redundant protein structures, both at the level of an individual protein and at the level of structural domains. In this investigation, we were able to separately quantify the self-similar symmetries in spatial correlation patterns amongst peptide–dipole units, charged amino acids, residues with the π-electron cloud and hydrophobic amino acids. The results revealed that electrostatic environments in the interiors of proteins belonging to ‘α/α toroid’ (all-α class) and ‘PLP-dependent transferase-like’ domains (α/β class) are highly conducive. In contrast, the interiors of ‘zinc finger design’ (‘designed proteins’) and ‘knottins’ (‘small proteins’) were identified as folds with the least conducive electrostatic environments. The fold ‘conotoxins’ (peptides) could be unambiguously identified as one type with the least stability. The same analyses revealed that peptide–dipoles in the α/β class of proteins, in general, are more correlated to each other than are the peptide–dipoles in proteins belonging to the all-α class. Highly favorable electrostatic milieu in the interiors of TIM-barrel, α/β-hydrolase structures could explain their remarkably conserved (evolutionary) stability from a new light. Finally, we point out certain inherent limitations of fractal constructs before attempting to identify the areas and problems where the implementation of fractal dimension-based constructs can be of paramount help to unearth latent information on protein structural properties.     

Molybdenum cofactor biosynthesis and deficiency

The molybdenum cofactor (Moco) forms the active site of all molybdenum (Mo) enzymes, except nitrogenase. Mo enzymes catalyze important redox reactions in global metabolic cycles. Moco consists of Mo covalently bound to one or two dithiolates attached to a unique tricyclic pterin moiety commonly referred to as molybdopterin (MPT). Moco is synthesized by an ancient and conserved biosynthetic pathway that can be divided into four steps, according to the biosynthetic intermediates precursor Z (cyclic pyranopterin monophosphate), MPT and adenylated MPT. In a fifth step modifications such as attachment of nucleotides, sulfuration or bond formation between Mo and the protein result in different catalytic Mo centers. A defect in any of the steps of Moco biosynthesis results in the pleiotropic loss of all Mo enzyme activities. Human Moco deficiency is a hereditary metabolic disorder characterized by severe neurodegeneration resulting in early childhood death. Recently, a first substitution therapy was established. Received 17 June 2005; received after revision 18 August 2005; accepted 1 September 2005     

Protein kinase CK2 and new binding partners during spermatogenesis

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α′-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α′, TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.     

Small heat shock proteins: molecular structure and chaperone function

Small heat shock proteins (sHSPs) associate with nuclei, cytoskeleton and membranes, and as molecular chaperones they bind partially denatured proteins, thereby preventing irreversible protein aggregation during stress. sHSP monomers consist of a conserved α-crystallin domain of approximately 90 amino acid residues, bordered by variable amino- and carboxy-terminal extensions. The sHSPs undergo dynamic assembly into mono- and poly-disperse oligomers where the rate of disassembly affects chaperoning. The α-crystallin domain contains several β-strands organized into two β-sheets responsible for dimer formation, the basic building block of most sHSPS. The amino-terminal extension modulates oligomerization, subunit dynamics and substrate binding, whereas the flexible carboxy-terminal extension promotes solubility, chaperoning and oligomerization, the latter by inter-subunit linkage. Crystallization studies have revealed sHSP structure and function. Additionally, site-directed mutagenesis, biophysical investigations, functional studies and the discovery of relationships between mutated sHSPs and diseases have illuminated the role of sHSP within cells. Received 8 May 2005; received after revision 24 June 2005; accepted 19 July 2005     

Structural insights into the multiple functions of protein C inhibitor

Protein C inhibitor (PCI) is a widely distributed, multifunctional member of the serpin family of protease inhibitors, and has been implicated in several physiological processes and disease states. Its inhibitory activity and specificity are regulated by binding to cofactors such as heparin, thrombomodulin and phospholipids, and it also appears to have non-inhibitory functions related to hormone and lipid binding. Just how the highly conserved serpin architecture can support the multiple diverse functions of PCI is a riddle best addressed by protein crystallography. Over the last few years we have solved the structure of PCI in its native, cleaved and protein-complexed states. They reveal a conserved serpin fold and general mechanism of protease inhibition, but with some unique features relating to inhibitory specificity/promiscuity, cofactor binding and hydrophobic ligand transport. Received 1 July 2008; received after revision 16 August 2008; accepted 22 August 2008     

��-Glucosidases

β-Glucosidases (3.2.1.21) are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. β-Glucosidases function in glycolipid and exogenous glycoside metabolism in animals, defense, cell wall lignification, cell wall β-glucan turnover, phytohormone activation, and release of aromatic compounds in plants, and biomass conversion in microorganisms. These functions lead to many agricultural and industrial applications. β-Glucosidases have been classified into glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, and GH30, based on their amino acid sequences, while other β-glucosidases remain to be classified. The GH1, GH5, and GH30 β-glucosidases fall in GH Clan A, which consists of proteins with (β/α)₈-barrel structures. In contrast, the active site of GH3 enzymes comprises two domains, while GH9 enzymes have (α/α)₆ barrel structures. The mechanism by which GH1 enzymes recognize and hydrolyze substrates with different specificities remains an area of intense study.     

The interaction of superoxide with nitric oxide destabilizes hypoxia-inducible factor-1α

In renal carcinoma cells (RCC4) hypoxia inducible factor-1 (HIF-1) is constitutively expressed due to a von Hippel Lindau protein deficiency, but can be degraded by calpain, independently of the 26S proteasome, when exposed to hypoxia/nitric oxide (NO). In this study we examined molecular mechanisms to explain calpain activation. The inability of hypoxia/NO to degrade HIF-1α in respiratory-deficient RCC4-ρ0 cells pointed to the requirement for mitochondria-derived reactive oxygen species. A prerequisite for O ₂ ⁻ in combination with NO to destabilize HIF-1α was corroborated in RCC4-p0 cells, when the redox cycler 2,3-dimethoxy-1,4-naphthoquinone was used as a source of superoxide. Degradation of HIF-1α required intracellular calcium transients and calpain activation. Using uric acid to interfere with signal transmission elicited by NO/O ₂ ⁻ blocked HIF-1α degradation and attenuated a calcium increase. We conclude that an oxidative signal as a result of NO/O ₂ ⁻ coformation triggers a calcium increase that activates calpain to degrade HIF-1α, independently of the proteasome. Received 14 August 2007; received after revision 4 October 2007; accepted 22 October 2007     

Three-dimensional structure of annexins

Annexins constitute a family of structurally related calcium- and phospholipid-binding proteins whose molecular structure has been investigated in detail in the crystalline and membrane-bound form. Their polypeptide chain is folded into four or eight α-helical domains of similar structure with a central hydrophilic pore. Bound to phospholipid membranes, the four-domain arrangement of the annexin molecule is conserved. A peripheral binding mode has been well documented by electron microscopy and a variety of other techniques.     

Novel role of cPLA2α in membrane and actin dynamics

Actin-directed processes such as membrane ruffling and cell migration are regulated by specific signal transduction pathways that become activated by growth factor receptors. The same signaling pathways that lead to modifications in actin dynamics also activate cPLA₂α. Moreover, arachidonic acid, the product of cPLA₂α activity, is involved in regulation of actin dynamics. Therefore, it was investigated whether cPLA₂α plays a role in actin dynamics, more specifically during growth factor-induced membrane ruffling and cell migration. Upon stimulation of ruffling and cell migration by growth factors, endogenous cPLA₂α and its active phosphorylated form were shown to relocate at protrusions of the cell membrane involved in actin and membrane dynamics. Inhibition of cPLA₂α activity with specific inhibitors blocked growth factor-induced membrane and actin dynamics, suggesting an important role for cPLA₂α in these processes.     

nDsbD: a redox interaction hub in the Escherichia coli periplasm

DsbD is a redox-active protein of the inner Escherichia coli membrane possessing an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain. nDsbD interacts with four different redox proteins involved in the periplasmic disulfide isomerization and in the cytochrome c maturation systems. We review here the studies that led to the structural characterization of all soluble DsbD domains involved and, most importantly, of trapped disulfide intermediate complexes of nDsbD with three of its four redox partners. These results revealed the structural features enabling nDsbD, a ‘redox hub’ with an immunoglobulin-like fold, to interact efficiently with its different thioredoxin-like partners. Received 3 February 2006; received after revision 1 March 2006; accepted 5 April 2006     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p₁ ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p₂ and p₀ have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p₂ , and Lys-66 in p₀ ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p₂ are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p₀ electrostatic interaction contributes only to binding of the RNA.     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998