Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

Calcitonin gene‐related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor‐like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP‐CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1‐CLR ECD fusion purified as a monomer, whereas the RAMP2‐CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27‐37) and AM(37‐52) fragments were identified as the minimal ECD complex binding regions. The CGRP C‐terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C‐terminal amide group was essential for ECD binding. Alanine‐scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.     

Construction of pH‐sensitive Her2‐binding IgG1‐Fc by directed evolution

For most therapeutic proteins, a long serum half‐life is desired. Studies have shown that decreased antigen binding at acidic pH can increase serum half‐life. In this study, we aimed to investigate whether pH‐dependent binding sites can be introduced into antigen binding crystallizable fragments of immunoglobulin G1 (Fcab). The C‐terminal structural loops of an Fcab were engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2‐ECD) at pH 6 compared to pH 7.4. A yeast‐displayed Fcab‐library was alternately selected for binding at pH 7.4 and non‐binding at pH 6.0. Selected Fcab variants showed clear pH‐dependent binding to soluble Her2‐ECD (decrease in affinity at pH 6.0 compared to pH 7.4) when displayed on yeast. Additionally, some solubly expressed variants exhibited pH‐dependent interactions with Her2‐positive cells whereas their conformational and thermal stability was pH‐independent. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. The study demonstrates that yeast surface display is a valuable tool for directed evolution of pH‐dependent binding sites in proteins.     

Structural studies of the natriuretic peptide receptor: a novel hormone-induced rotation mechanism for transmembrane signal transduction

The atrial natriuretic peptide (ANP) receptor is a single-span transmembrane receptor that is coupled to its intrinsic intracellular guanylate cyclase (GCase) catalytic activity. To investigate the mechanisms of hormone binding and signal transduction, we have expressed the extracellular hormone-binding domain of the ANP receptor (ANPR) and characterized its structure and function. The disulfide-bond structure, state of glycosylation, binding-site residues, chloride-dependence of ANP binding, dimerization, and binding stoichiometry have been determined. More recently, the crystal structures of both the apoANPR dimer and ANP-bound complex have been determined. The structural comparison between the two has shown that, upon ANP binding, two ANPR molecules in the dimer undergo an inter-molecular twist with little intra-molecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains with essentially no change in the inter-domain distance. This movement alters the relative orientation of the two domains equivalent to counter-clockwise rotation of each by 24 degrees . These results suggest that transmembrane signaling by the ANP receptor is mediated by a novel hormone-induced rotation mechanism.     

Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro

Miro is a highly conserved calcium‐binding GTPase at the regulatory nexus of mitochondrial transport and autophagy. Here we present crystal structures comprising the tandem EF hand and carboxy terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified ‘hidden’ EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide‐sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand–cGTPase interface. Our results indicate structural mechanisms for calcium, nucleotide and phosphorylation‐dependent regulation of mitochondrial function by Miro.     

The importance of valine 114 in ligand binding in β2‐adrenergic receptor

G‐protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. β₂‐Adrenergic receptor (β₂‐AR) is a well‐studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand‐binding region of β₂‐AR using the recently solved high‐resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure‐function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster β₂‐AR by site‐directed mutagenesis. We replaced V114 in hamster β₂‐AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G‐protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side‐chains but also the aryl‐ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in β₂‐AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.     

Crystal structure and site 1 binding energetics of human placental lactogen

In primates, placental lactogen (PL) is a pituitary hormone with fundamental roles during pregnancy involving fetal growth, metabolism, and stimulating lactation in the mother. Human placental lactogen (hPL) is highly conserved with human growth hormone (hGH) and both hormones bind to the hPRLR extracellular domain (ECD), the first step in receptor homodimerization, in a Zn2+-dependent manner. A modified surface plasmon resonance method was developed to measure the kinetics for hPL and hGH binding to the hPRLR ECD, with and without Zn2+ and showed that hPL has about a tenfold higher affinity for the hPRLR ECD1 than hGH. The crystal structure of the free state of hPL has been determined to 2.0 A resolution showing the molecule possesses an overall structure similar to other long chain four-helix bundle cytokines. Comparison of the free hPL structure with the 1:1 complex structure of hGH bound to the hPRLR ECD1 suggests that two surface loops undergo conformational changes >10 A upon binding. An 18 residue Ala-scan was used to characterize the binding energy epitope for the site 1 interface of hPL. Individual alanine substitutions at five positions reduced binding affinity by a DeltaDeltaG > or = 3 kcal mol(-1). A comparison of the hPL site 1 epitope with that previously determined for hGH indicates contributions of individual residues track reasonably well between hPL and hGH. In particular, residues involved in the zinc-binding site and Lys172 constitute the principal binding determinants for both hormones. However, several residues that are identical between hPL and hGH contribute quite differently to the binding of the hPRLR ECD1. Additionally, the overall magnitudes of the DeltaDeltaG changes observed from the Ala-scan of hPL were markedly larger than those determined in the comparative scan of hGH to the hPRLR ECD1. The structural and biophysical data presented here show that subtle changes in the structural context of an interaction can lead to significantly different effects at the individual residue level.     

Recognition of an intra‐chain tandem 14‐3‐3 binding site within PKCε

The phosphoserine/threonine binding protein 14‐3‐3 stimulates the catalytic activity of protein kinase C‐ε (PKCε) by engaging two tandem phosphoserine‐containing motifs located between the PKCε regulatory and catalytic domains (V3 region). Interaction between 14‐3‐3 and this region of PKCε is essential for the completion of cytokinesis. Here, we report the crystal structure of 14‐3‐3ζ bound to a synthetic diphosphorylated PKCε V3 region revealing how a consensus 14‐3‐3 site and a divergent 14‐3‐3 site cooperate to bind to 14‐3‐3 and so activate PKCε. Thermodynamic data show a markedly enhanced binding affinity for two‐site phosphopeptides over single‐site 14‐3‐3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14‐3‐3 ligand. This dual‐site intra‐chain recognition has implications for other 14‐3‐3 targets, which seem to have only a single 14‐3‐3 motif, as other lower affinity and cryptic 14‐3‐3 gatekeeper sites might exist.     

Crystal structure of the trimeric N‐terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions

Centrin is a member of the EF‐hand superfamily of calcium‐binding proteins, a highly conserved eukaryotic protein that binds to Ca²⁺. Its self‐assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N‐terminal domain of ciliate Euplotes octocarinatus centrin (N‐EoCen) was determined by using the selenomethionine single‐wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca²⁺ ion‐binding sites I and II, protomer A, and C bound one Ca²⁺ ion, while protomer B bound two Ca²⁺ ions. A novel binding site III was observed and the Ca²⁺ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.     

Thermodynamics of protein‐cation interaction: Ca+2 and Mg+2 binding to the fifth binding module of the LDL receptor

The ligand binding domain of the LDL receptor (LDLR) contains seven structurally homologous repeats. The fifth repeat (LR5) is considered to be the main module responsible for the binding of lipoproteins LDL and β‐VLDL. LR5, like the other homologous repeats, is around 40‐residue long and contains three disulfide bonds and a conserved cluster of negatively charged residues surrounding a hexacoordinated calcium ion. The calcium coordinating cage is formed by the backbone oxygens of W193 and D198, and side‐chain atoms of D196, D200, D206, and E207. The functionality of LDLR is closely associated with the presence of calcium. Magnesium ions are to some extent similar to calcium ions. However, they appear to be involved in different physiological events and their concentrations in extracellular and intracellular compartments are regulated by different mechanisms. Whether magnesium ions can play a role in the complex cycle of LDLR internalization and recycling is not known. We report here a detailed study of the interaction between LR5 and these two cations combining ITC, emission fluorescence, high resolution NMR, and MD simulations, at extracellular and endosomal pHs. Our results indicate that the conformational stability and internal dynamics of LR5 are strongly modulated by the specific bound cation. It appears that the difference in binding affinity for these cations is somewhat compensated by their different concentrations in late LDL‐associated endosomes. While the mildly acidic and calcium‐depleted environment in late endosomes has been proposed to contribute significantly to LDL release, the presence of magnesium might assist in efficient LDLR recycling. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Nucleotides sequestered at different subsite loci within DNA‐binding pockets of two OB‐fold single‐stranded DNA‐binding proteins are unstacked to different extents

The gene 5 protein (g5p) encoded by the Ff strains of Escherichia coli bacteriophages is a dimeric single‐stranded DNA‐binding protein (SSB) that consists of two identical OB‐fold (oligonucleotide/oligosaccharide‐binding) motifs. Ultrafast time‐resolved fluorescence measurements were carried out to investigate the effect of g5p binding on the conformation of 2‐aminopurine (2AP) labels positioned between adenines or cytosines in the 16‐nucleotide antiparallel tails of DNA hairpins. The measurements revealed significant changes in the conformational heterogeneity of the 2AP labels caused by g5p binding. The extent of the changes was dependent on sub‐binding‐site location, but generally resulted in base unstacking. When bound by g5p, the unstacked 2AP population increased from ～22% to 59–67% in C‐2AP‐C segments and from 39% to 77% in an A‐2AP‐A segment. The OB‐fold RPA70A domain of the human replication protein A also caused a significant amount of base unstacking at various locations within the DNA binding site as evidenced by steady‐state fluorescence titration measurements using 2AP‐labeled 5‐mer DNAs. These solution studies support the concept that base unstacking at most of a protein's multiple sub‐binding‐site loci may be a feature that allows non‐sequence specific OB‐fold proteins to bind to single‐stranded DNAs (ssDNAs) with minimal preference for particular sequences. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 484–496, 2013.     

Prediction of protein‐glucose binding sites using support vector machines

Glucose is a simple sugar that plays an essential role in many basic metabolic and signaling pathways. Many proteins have binding sites that are highly specific to glucose. The exponential increase of genomic data has revealed the identity of many proteins that seem to be central to biological processes, but whose exact functions are unknown. Many of these proteins seem to be associated with disease processes. Being able to predict glucose‐specific binding sites in these proteins will greatly enhance our ability to annotate protein function and may significantly contribute to drug design. We hereby present the first glucose‐binding site classifier algorithm. We consider the sugar‐binding pocket as a spherical spatio‐chemical environment and represent it as a vector of geometric and chemical features. We then perform Random Forests feature selection to identify key features and analyze them using support vector machines classification. Our work shows that glucose binding sites can be modeled effectively using a limited number of basic chemical and residue features. Using a leave‐one‐out cross‐validation method, our classifier achieves a 8.11% error, a 89.66% sensitivity and a 93.33% specificity over our dataset. From a biochemical perspective, our results support the relevance of ordered water molecules and ions in determining glucose specificity. They also reveal the importance of carboxylate residues in glucose binding and the high concentration of negatively charged atoms in direct contact with the bound glucose molecule. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Arg‐85 and Thr‐430 in murine 5‐aminolevulinate synthase coordinate acyl‐CoA‐binding and contribute to substrate specificity

5‐Aminolevulinate synthase (ALAS) controls the rate‐limiting step of heme biosynthesis in mammals by catalyzing the condensation of succinyl‐coenzyme A and glycine to produce 5‐aminolevulinate, coenzyme‐A (CoA), and carbon dioxide. ALAS is a member of the α‐oxoamine synthase family of pyridoxal 5′‐phosphate (PLP)‐dependent enzymes and shares high degree of structural similarity and reaction mechanism with the other members of the family. The X‐ray crystal structure of ALAS from Rhodobacter capsulatus reveals that the alkanoate component of succinyl‐CoA is coordinated by a conserved arginine and a threonine. The functions of the corresponding acyl‐CoA‐binding residues in murine erthyroid ALAS (R85 and T430) in relation to acyl‐CoA binding and substrate discrimination were examined using site‐directed mutagenesis and a series of CoA‐derivatives. The catalytic efficiency of the R85L variant with octanoyl‐CoA was 66‐fold higher than that of the wild‐type protein, supporting the proposal of this residue as key in discriminating substrate binding. Substitution of the acyl‐CoA‐binding residues with hydrophobic amino acids caused a ligand‐induced negative dichroic band at 420 nm in the CD spectra, suggesting that these residues affect substrate‐mediated changes to the PLP microenvironment. Transient kinetic analyses of the R85K variant‐catalyzed reactions confirm that this substitution decreases microscopic rates associated with formation and decay of a key reaction intermediate and show that the nature of the acyl‐CoA tail seriously affect product binding. These results show that the bifurcate interaction of the carboxylate moiety of succinyl‐CoA with R85 and T430 is an important determinant in ALAS function and may play a role in substrate specificity.     

Conformational plasticity of the calcium‐binding pocket in the Burkholderia glumae lipase: Remodeling induced by mutation of calcium coordinating residues

Most bacterial lipases bind one or more Ca²⁺ atoms at different locations and are a suitable case of study for investigating structural effects related to calcium binding, depletion, or mutation of calcium‐binding sites. Generally Ca²⁺ in microbial lipases can play a crucial role in the stabilization of the whole three‐dimensional structure by mediating long‐range effects. It has been recently demonstrated that calcium binding influences thermal stability of Burkholderia glumae lipase (BGL) through the restriction of conformational plasticity of specific regions. Moreover, calcium depletion results in a highly cooperative protein unfolding, eliciting protein aggregation. To further shed light on molecular mechanisms and structural features connected to calcium binding in microbial lipases, we present a molecular dynamics investigation, based on multiple‐replica approach at different temperatures, of BGL mutants targeting the calcium‐binding site. It turns out that additional acidic residues, which are conserved in other microbial lipases, help in overcoming effects induced by mutation of D241 Ca²⁺‐coordinating residue, upon rearrangements induced in the calcium binding site. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 117–126, 2011.     

Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors

Membrane bound guanylyl cyclases are single chain transmembrane receptors that produce the second messenger cGMP by either intra- or extracellular stimuli. This class of type I receptors contain an intracellular catalytic guanylyl cyclase domain, an adjacent kinase-like domain and an extracellular ligand binding domain though some receptors have their ligands yet to be identified. The most studied member is the atrial natriuretic peptide (ANP) receptor, which is involved in blood pressure regulation. Extracellular ANP binding induces a conformational change thereby activating the pre-oligomerized receptor leading to the production of cGMP. The recent crystal structure of the dimerized hormone binding domain of the ANP receptor provides a first three-dimensional view of this domain and can serve as a basis to structurally analyze mutagenesis, cross-linking, and genetic studies of this class of receptors as well as a non-catalytic homolog, the clearance receptor. The fold of the ligand binding domain is that of a bilobal periplasmic binding protein (PBP) very similar to that of the Leu/Ile/Val binding protein, AmiC, multi-domain transmembrane metabotropic glutamate receptors, and several DNA binding proteins such as the lactose repressor. Unlike these structural homologs, the guanylyl cyclase receptors bind much larger molecules at a site seemingly remote from the usual small molecule binding site in periplasmic binding protein folds. Detailed comparisons with these structural homologs offer insights into mechanisms of signal transduction and allosteric regulation, and into the remarkable usage of the periplasmic binding protein fold in multi-domain receptors/proteins.     

Cooperative DNA‐binding and sequence‐recognition mechanism of aristaless and clawless

To achieve accurate gene regulation, some homeodomain proteins bind cooperatively to DNA to increase those site specificities. We report a ternary complex structure containing two homeodomain proteins, aristaless (Al) and clawless (Cll), bound to DNA. Our results show that the extended conserved sequences of the Cll homeodomain are indispensable to cooperative DNA binding. In the Al–Cll–DNA complex structure, the residues in the extended regions are used not only for the intermolecular contacts between the two homeodomain proteins but also for the sequence‐recognition mechanism of DNA by direct interactions. The residues in the extended N‐terminal arm lie within the minor groove of DNA to form direct interactions with bases, whereas the extended conserved region of the C‐terminus of the homeodomain interacts with Al to stabilize and localize the third α helix of the Cll homeodomain. This structure suggests a novel mode for the cooperativity of homeodomain proteins.