Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis

Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.     

Structural characterization of the caveolin scaffolding domain in association with cholesterol-rich membranes

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a β-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect β-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.     

Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.     

A molecular dissection of caveolin-1 membrane attachment and oligomerization. Two separate regions of the caveolin-1 C-terminal domain mediate membrane binding and oligomer/oligomer interactions in vivo

Caveolins form interlocking networks on the cytoplasmic face of caveolae. The cytoplasmically directed N and C termini of caveolins are separated by a central hydrophobic segment, which is believed to form a hairpin within the membrane. Here, we report that the caveolin scaffolding domain (CSD, residues 82-101), and the C terminus (residues 135-178) of caveolin-1 are each sufficient to anchor green fluorescent protein (GFP) to membranes in vivo. We also show that the first 16 residues of the C terminus (i.e. residues 135-150) are necessary and sufficient to attach GFP to membranes. When fused to the caveolin-1 C terminus, GFP co-localizes with two trans-Golgi markers and is excluded from caveolae. In contrast, the CSD targets GFP to caveolae, albeit less efficiently than full-length caveolin-1. Thus, caveolin-1 contains at least two membrane attachment signals: the CSD, dictating caveolar localization, and the C terminus, driving trans-Golgi localization. Additionally, we find that caveolin-1 oligomer/oligomer interactions require the distal third of the caveolin-1 C terminus. Thus, the caveolin-1 C-terminal domain has two separate functions: (i) membrane attachment (proximal third) and (ii) protein/protein interactions (distal third).     

Sequence and structural diversity in endotoxin-binding dodecapeptides

For the study of sequence or structure requirement of short peptides for endotoxin binding, and to search for potential endotoxin antagonists, biopanning was carried out on a phage-displayed random dodecapeptide library against immobilized lipopolysaccharide (LPS) or lipid A (LA), the core toxic portion of LPS. Specific binding of selected phage-displayed peptides to LPS/LA was confirmed by surface plasmon resonance (SPR) analysis. These peptides are rich in basic and hydrophobic amino acids, especially histidine, proline and tryptophan, highlighting apparent amphiphilicity and bacterial membrane activity. These dodecapeptide sequences have no predictable secondary structure in solution, indicating the importance of a random structure before their interaction with LPS/LA. Sequence alignment reveals various potential secondary structures with these selected peptides, which contain specific signature motifs such as b(p)hb(p)hb(p), bbbb, hhhh (b-basic, p-polar, h-hydrophobic residue), capable of binding LPS/LA. However, none of these peptides exhibit a significant calculated structural amphiphilicity while assuming a secondary structure. This study suggests that for these short dodecapeptides to bind LPS/LA, the potential for their structural adaptation is more important than an amphipathic structure.     

Oligomeric structure of the chemokine CCL5/RANTES from NMR, MS, and SAXS data

CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in?vitro, CCL5's propensity for aggregation is essential for migration in?vivo, T?cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in?vivo.     

Characterization of the highly divergent U2 RNA homolog in the microsporidian Vairimorpha necatrix.

An RNA homologous to U2 RNA and a single copy gene encoding the RNA homolog have been characterized in the microsporidian, Vairimorpha necatrix. The RNA which is 165 nucleotides in length possesses significant similarity to U2 RNA, particularly in the 5' half of the molecule. The U2 homolog contains the highly conserved GUAGUA branch point binding sequence seen in all U2 RNAs except those of the trypanosomes. A U2 RNA sequence element implicated in a U2:U6 RNA intermolecular pairing is also present in the U2 homolog. The V. necatrix U2 RNA homolog differs at positions previously found to be invariant in U2 RNAs and appears to lack an Sm binding site sequence. The RNA can be folded into a secondary structure possessing three of the four principal stem-loops proposed for the consensus U2 RNA structure. A cis-diol containing cap structure is present at the 5' end of the U2 homolog. Unlike the cap structures seen in U-snRNAs and mRNAs it is neither 2,2,7-trimethylguanosine, gamma-monomethyl phosphate, nor 7-methylguanosine.     

On the DNA binding protein II from Bacillus stearothermophilus. I. Purification, studies in solution, and crystallization

DNA binding protein II from Bacillus stearothermophilus has been purified as a single species from the nonribosomal cell fraction by a combination of gel filtration and ion exchange chromatography. The protein occurs in solution as a tetramer and is able to bind to 30 S, 50 S, and 70 S ribosomal particles. Circular dichroism studies show that the protein has approximately 45% alpha-helix. The secondary structure of the Bacillus protein is considerably more resistant to the effects of increasing temperature and urea concentration than the homologous protein (NS1 and NS2) from Escherichia coli. Proton magnetic resonance experiments show that the protein has a well folded, compact tertiary structure. The DNA binding protein has been crystallized from several precipitants as monoclinic needles and triclinic plates. The monoclinic form diffracts to at least 3.5 A and oscillation data from the native crystals have been collected. The protein is able to bind to both single- and double-stranded oligodeoxyribonucleotides. Upon binding, several changes occur in the protein NMR spectrum which may be used for further analysis of the mechanism of interaction.     

NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

The emergence of drug‐resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X‐ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites.     

Structural divergence is more extensive than sequence divergence for a family of intrinsically disordered proteins

The p53 transactivation domain (p53TAD) is an intrinsically disordered protein (IDP) domain that undergoes coupled folding and binding when interacting with partner proteins like the E3 ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70. The secondary structure and dynamics of six closely related mammalian homologues of p53TAD were investigated using nuclear magnetic resonance (NMR) spectroscopy. Differences in both transient secondary structure and backbone dynamics were observed for the homologues. Many of these differences were localized to the binding sites for MDM2 and RPA70. The amount of transient helical secondary structure observed for the MDM2 binding site was lower for the dog and mouse homologues, compared with human, and the amount of transient helical secondary structure observed for the RPA70 binding site was higher for guinea pig and rabbit, compared with human. Differences in the amount of transient helical secondary structure observed for the MDM2 binding site were directly related to amino acid substitutions occurring on the solvent exposed side of the amphipathic helix that forms during the p53TAD/MDM2 interaction. Differences in the amount of transient helical secondary structure were not as easily explained for the RPA70 binding site because of its extensive sequence divergence. Clustering analysis shows that the divergence in the transient secondary structure of the p53TAD homologues exceeds the amino acid sequence divergence. In contrast, strong correlations were observed between the backbone dynamics of the homologues and the sequence identity matrix, suggesting that the dynamic behavior of IDPs is a conserved evolutionary feature. Proteins 2013; 81:1686–1698. © 2013 Wiley Periodicals, Inc.     

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA

The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.     

Combinatorial alanine-scanning

Combinatorial libraries of alanine-substituted proteins can be used to rapidly identify residues important for protein function, stability and shape. Each alanine substitution examines the contribution of an individual amino acid sidechain to the functionality of the protein. The recently described method of shotgun scanning uses phage-displayed libraries of alanine-substituted proteins for high-throughput analysis.     

The functional binding epitope of a high affinity variant of human growth hormone mapped by shotgun alanine-scanning mutagenesis: insights into the mechanisms responsible for improved affinity

A high-affinity variant of human growth hormone (hGH(v)) contains 15 mutations within site 1 and binds to the hGH receptor (hGHR) approximately 400-fold tighter than does wild-type (wt) hGH (hGH(wt)). We used shotgun scanning combinatorial mutagenesis to dissect the energetic contributions of individual residues within the hGH(v) binding epitope and placed them in context with previously determined structural information. In all, the effects of alanine substitutions were determined for 35 hGH(v) residues that are directly contained in or closely border the binding interface. We found that the distribution of binding energy in the functional epitope of hGH(v) differs significantly from that of hGH(wt). The residues that contributed the majority of the binding energy in the wt interaction (the so-called binding "hot spot") remain important, but their contributions are attenuated in the hGH(v) interaction, and additional binding energy is acquired from residues on the periphery of the original hotspot. Many interactions that inhibited the binding of hGH(wt) are replaced by interactions that make positive contributions to the binding of hGH(v). These changes produce an expanded and diffused hot spot in which improved affinity results from numerous small contributions distributed broadly over the interface. The mutagenesis results are consistent with previous structural studies, which revealed widespread structural differences between the wt and variant hormone-receptor interfaces. Thus, it appears that the improved binding affinity of hGH(v) site 1 was not achieved through minor adjustments to the wt interface, but rather, results from a wholesale reconfiguration of many of the original binding elements.     

Characterization the Effects of Structure and Energetics of Intermolecular Interactions on the Oligomerization of Peptides in Aqueous 2, 2, 2-Trifluoroethanol via Circular Dichroism and Nuclear Magnetic Resonance Spectroscopy

Intermolecular interactions are of fundamental importance to fully comprehend a wide range of protein behaviors such as oligomerization, folding and recognition. Two peptides, NPY^[18−36] and NPY^[15−29], segmented from human neuropeptide Y (hNPY), were synthesized in this work to study the interaction between species. Information about intermolecular interactions was extracted from their oligomerizing behaviors. The results from CD and NMR showed that the addition of 2, 2, 2-trifluoroethanol (TFE) induces a stable helix in each peptides and an extended helix in NPY^[18−36], formed between residues 30-36. Pulsed field gradient NMR data revealed that NPY^[15−29] forms a larger oligomer at lower temperatures and continuously dissociates into the monomeric form with increasing temperature. NPY^[18−36] was also found to undergo an enhanced interaction with TFE and a more favorable self-association at higher temperatures. We characterized the changes of oligomerized states with respect to temperature to infer the effects of entropy and interaction energy on the association-dissociation equilibrium. As shown by NPY^[15−29], deletion of helical secondary structure or residues from the C-terminal segment may disrupt the solvation by TFE and results in entropy increase as the oligomer dissociates. Unlike that in NPY^[15−29], the extended helix in NPY^[18−36] improves the binding of TFE, and as a result, entropy is gained via the transfer of the TFE cluster from the interface between monomeric peptides into the bulk solvent. This observation suggests that the oligomerized state may be modulated by the entropy and energetics contributed by helical segments in the oligomerization process.     

The caveolin scaffolding domain modifies 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor binding properties by inhibiting phospholipase A2 activity

Activation of the enzyme phospholipase (PLA 2) has been proposed to be part of the molecular mechanism involved in the alteration of 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor responsiveness during long term changes in synaptic plasticity (long term potentiation). This study assesses the effect of the caveolin-1 scaffolding domain (CSD) on the activity of the regulatory enzyme PLA2. Caveolin-1 is a 22-kDa cholesterol-binding membrane protein known to inhibit the activity of most of its interacting partners. Our results show that the calcium-dependent cytosolic form of PLA2 (cPLA2) and caveolin-1 co-localized in mouse primary hippocampal neuron cultures and that they were co-immunoprecipitated from mouse hippocampal homogenates. A peptide corresponding to the scaffolding domain of caveolin-1 (Cav-(82-101)) dramatically inhibited cPLA2 activity in purified hippocampal synaptoneurosomes. Activation of endogenous PLA2 activity with KCl or melittin increased the binding of [3H]AMPA to its receptor. This effect was almost completely abolished by the addition of the CSD peptide to these preparations. Moreover, we demonstrated that the inhibitory action of the CSD peptide on AMPA receptor binding properties is specific (because a scrambled version of this peptide failed to have any effect) and that it is mediated by an inhibition of PLA2 enzymatic activity (because the CSD peptide failed to have an effect in membrane preparations lacking endogenous PLA2 activity). These results raised the possibility that caveolin-1, via the inhibition of cPLA2 enzymatic activity, may interfere with synaptic facilitation and long term potentiation formation in the hippocampus.     

The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the other (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they are the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.