Functional identification of calcium binding residues in bovine alpha-lactalbumin

The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.     

Kinetic analysis of a mutational hot spot in the EcoRV restriction endonuclease

The EcoRV endonuclease contacts the minor groove of DNA through a peptide loop encompassing residues 67-72. This loop adapts to distorted DNA in the specific complex and to regular DNA in the nonspecific complex. Random mutagenesis had previously identified glutamine 69 as the key component of the loop and this study reports on mutants with glutamate (Q69E), lysine (Q69K), or leucine (Q69L) at this position. The mutants bound DNA specifically at the EcoRV recognition site in the presence of Ca2+, in the same manner as wild-type EcoRV. In the absence of divalent metals, Q69K and Q69L showed the same nonspecific binding as native EcoRV while Q69E failed to bind DNA. Glutamate at position 69 presumably repels nonspecific DNA whilst allowing the adaptations to specific DNA. Both Q69E and Q69K had severely impaired DNA cleavage activities, while Q69L had a steady-state k(cat) within an order of magnitude of wild-type EcoRV though its primary product was nicked DNA, in contrast to double strand breaks by wild-type EcoRV. The activity of Q69L required higher concentrations of Mg2+ than the wild-type and showed a sigmoidal dependence upon the Mg2+ concentration, indicating two metal ions per strand scission. Transient kinetics on Q69L gave lower rate constants for phosphodiester hydrolysis than wild-type EcoRV and its reaction also involved a slow conformational change preceding DNA cleavage that had no equivalent with the wild-type. Gln69 in EcoRV thus plays key roles in the adjustments of the protein to varied DNA structures and in the alignment of the catalytic functions for DNA cleavage.     

Ikappa Balpha functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-kappaB

We have determined the binding energies of complexes formed between Ikappa Balpha and the wild type and mutational variants of three different Rel/NF-kappaB dimers, namely, the p50/p65 heterodimer and homodimers of p50 and p65. We show that although a common mode of interaction exists between the Rel/NF-kappaB dimers and Ikappa Balpha, IkappaB alpha binds the NF-kappaB p50/p65 heterodimer with 60- and 27-fold higher affinity than the p50 and p65 homodimers, respectively. Each of the three flexibly linked segments of the rel homology region of Rel/NF-kappaB proteins (the nuclear localization sequence, the dimerization domain, and the amino-terminal DNA binding domain) is directly engaged in forming the protein/protein interface with the ankyrin repeats and the carboxyl-terminal acidic tail/PEST sequence of Ikappa Balpha. In the cell, Ikappa Balpha functions to retain NF-kappaB in the cytoplasm and inhibit its DNA binding activity. These properties are a result of the direct involvement of the nuclear localization sequences and of the DNA binding region of NF-kappaB in complex with Ikappa Balpha. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-kappaB dimers and the ankyrin domains of related proteins.     

Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site

Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a secreted serpin identified in extracellular matrixes. We show that PEDF extractions from the interphotoreceptor matrix are more efficient with increasing NaCl concentrations, indicating that ionic interactions mediate its association with this polyanionic matrix. We have used affinity chromatography and ultrafiltration to probe for direct binding of PEDF to glycosaminoglycans/polyanions. Correctly folded PEDF bound to immobilized heparin, chondroitin sulfate-A, -B, -C, and dextran sulfate columns and eluted from each with an increase in NaCl concentration. However, in the presence of urea, the protein lost its affinity for heparin. Binding of PEDF to heparan sulfate proteoglycan in solution was in a concentration-dependent fashion (half-maximal specific binding EC50 = 40 micrograms/mL) and was sensitive to increasing NaCl concentrations. The glycosaminoglycan-binding region was analyzed using chemical modification and limited proteolysis. PEDF chemically modified on lysine residues by biotinylation lost its capacity for interacting with heparin, implicating the involvement of PEDF lysine residues in heparin binding. Cleavage of the serpin-exposed loop with chymotrypsin did not affect the heparin-binding property. A limited proteolysis product containing residues 21-approximately 260 bound to heparin with similar affinity as the intact PEDF. Homology modeling of PEDF based on the X-ray crystal structures of antithrombin III and ovalbumin shows a region at the center of beta-sheet A-strands 2 and 3- and helix F that has a basic electrostatic surface potential and is densely populated with lysines exposed to the surface (K134, K137, K189, K191, H212, and K214) that are available to interact with various glycosaminoglycans/polyanions. This region represents a novel site for glycosaminoglycan binding in a serpin, which in PEDF, is distinct and nonoverlapping from the PEDF neurotrophic active region.     

Promoter specificity determinants of T7 RNA polymerase

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position -8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at -10 and -11, and Q758 with the bp at -8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.     

Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.     

A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins

Bacteriophage T5 5'-->3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single cleavage assay for this enzyme which uses a 5'-overhanging hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15). The catalytic assay has been extended to three mutants of T5 5'-->3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.     

Comparison of the structures and the crystal contacts of trypanosomal triosephosphate isomerase in four different crystal forms

Triosephosphate isomerase (TIM) is a dimeric enzyme consisting of 2 identical subunits. Trypanosomal TIM can be crystallized in 4 different spacegroups: P2(1)2(1)2(1), C2(big cell), C2(small cell), and P1. The P1 crystal form only grows in the presence of 1.4 M DMSO; there are 2 DMSO binding sites per subunit. The structures have been refined at a resolution of 1.83 A, 2.10 A, 2.13 A, and 1.80 A, respectively. In the 4 different spacegroups the TIM subunit can be observed in the context of 7 different crystallographic environments. In the C2 cells, the dimer 2-fold axis coincides with a crystallographic 2-fold axis. The similarities and differences of the 7 subunits are discussed. In 6 subunits the flexible loop (loop 6) is open, whereas in the P2(1)2(1)2(1) cell, the flexible loop of subunit 2 is in an almost closed conformation. The crystal contacts in the 4 different crystal forms are predominantly generated by polar residues in loops. A statistical analysis of the residues involved in crystal contacts shows that, in particular, serines are frequently involved in these interactions; 19% of the exposed serines are involved in crystal contacts.     

Solution dynamics and secondary structure of murine leukemia inhibitory factor: a four-helix cytokine with a rigid CD loop

Leukemia inhibitory factor (LIF) is a hematopoietic cytokine which elicits its effects on diverse cell types via both gp130 and a more specific LIF receptor. Recombinant murine LIF was studied by multidimensional homonuclear and 1H-15N heteronuclear NMR and 95% of backbone amide resonances assigned. Definition of the secondary structure by chemical shift data and NOE connectivities shows a four-alpha-helix bundle fold (helices A-D) in solution, with an additional flexible turn of helix in the AB loop. Subtle differences are seen in the conformations of helices A and D from those defined in the crystal structure [Robinson, R. C., Grey, L. M., Staunton, D., Vankelcom, H., Vernallis, A. B., Moreau, J.-F., Stuart, D. I., Heath, J. K., & Jones, E. Y. (1994) Cell77, 1101-1116]. The dynamics of the polypeptide backbone of LIF were assessed from 15N T1 and T2 relaxation times and 15N-1H heteronuclear NOEs of the amide groups. Using model-free formalism, the overall rotational correlation time of LIF in solution is calculated to be 9.7 ps. The four alpha-helices are relatively rigid, and high mobility is observed for N-terminal residues (Ser 1-Asn 21) and the AB loop. In contrast to several closely related cytokines, the long CD loop is relatively rigid. This may have implications for interactions with the specific LIF receptor, which binds in this region.     

Stress and strain in staphylococcal nuclease

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Functional interaction of an arginine conserved in the sixteen amino acid insertion module of Escherichia coli methionyl-tRNA formyltransferase with determinants for formylation in the initiator tRNA

Formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in eubacteria. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Previous studies suggested that a 16 amino acid insertion loop, present in all eubacterial MTF's (residues 34-49 in the E. coli enzyme), plays an important role in specific recognition of the initiator tRNA. Here, we have analyzed the effect of site-specific mutations of amino acids within this region. We show that an invariant arginine at position 42 within the loop plays a very important role both in the steps of substrate binding and in catalysis. The kinetic parameters of the R42K and R42L mutant enzymes using acceptor stem mutant initiator tRNAs as substrates suggest that arginine 42 makes functional contacts with the determinants at the 3:70 and possibly also the 2:71 base pairs in the acceptor stem of the initiator tRNA. The kinetic parameters of the G41R/R42L double mutant enzyme are essentially the same as those of R42L mutant, suggesting that the requirement for arginine at position 42 cannot be fulfilled by an arginine at position 41. Along with other data, this result suggests that the insertion loop, which is normally unstructured and flexible, adopts a defined conformation upon binding to the tRNA.