Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins.     

Prediction of loop regions in protein sequence

We suggest an algorithm that inputs a protein sequence and outputs a decomposition of the protein chain into a regular part including secondary structures and a nonregular part corresponding to loop regions. We have analyzed loop regions in a protein dataset of 3,769 globular domains and defined the optimal parameters for this prediction: the threshold between regular and nonregular regions and the optimal window size for averaging procedures using the scale of the expected number of contacts in a globular state and entropy scale as the number of degrees of freedom for the angles phi, psi, and chi for each amino acid. Comparison with known methods demonstrates that our method gives the same results as the well-known ALB method based on physical properties of amino acids (the percentage of true predictions is 64% against 66%), and worse prediction for regular and nonregular regions than PSIPRED (Protein Structure Prediction Server) without alignment of homologous proteins (the percentage of true predictions is 73%). The potential advantage of the suggested approach is that the predicted set of loops can be used to find patterns of rigid and flexible loops as possible candidates to play a structure/function role as well as a role of antigenic determinants.     

Prediction of natively unfolded regions in protein chains

Analysis showed that the globular or natively unfolded state of a protein can be inferred not only from a lower hydrophobicity or a higher charge, but also from the average environment density (average number of close residues located within a certain distance of a given one) of its residues. A database of 6626 protein structures was used to construct a statistical scale of the average number of close residues in globular structures for the 20 amino acids. The portion of false predictions in distinguishing between 80 globular and 90 natively unfolded proteins was 11% with the new scale and 17% with a hydrophobicity scale. The new scale proved suitable for predicting the folded or unfolded state for native proteins or the natively unfolded regions for protein chains. In comparisons with the available algorithms, the new method yielded the highest portion of true predictions (87 and 77% with averaging over residues and over proteins, respectively).     

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.     

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.     

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.     

Spelling protein structure

Recent sequence analysis of complete prokaryotic proteomes suggests that in early evolutionary stages proteins were rather small, of the size 25-35 amino acids. Corroborating evidence comes from protein crystal data, which indicate this size for closed loops--universal structural units of globular proteins. In the latest development we were able to derive and structurally characterize several sequence/structure prototypes apparently representing early protein units. Structurally the prototypes appear as closed loops stabilized by end-to-end van der Waals interactions. While nearly standard in size the loops are highly diverse in terms of their secondary structure. A presentation of the protein as an assembly of descendants of the prototypes, the first of its kind, is described in detail here. The sequence and structure of the ATP-binding subunit of histidine permease of S. typhimurium is shown to contain several modified copies of different prototype elements, closed loops, and, thus, can be spelled as: x-PI-x-PIV-PVI-PII-PVII-x, where PI-PVII are the prototype elements. This study sets up the basic principles for the sequence/structure prototype spelling of globular proteins.     

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.     

Predicting antibody hypervariable loop conformations. II: Minimization and molecular dynamics studies of MCPC603 from many randomly generated loop conformations

We describe a method for predicting the conformations of loops in proteins and its application to four of the complementarity determining regions [CDRs] in the crystallographically determined structure of MCPC603. The method is based on the generation of a large number of randomly generated conformations for the backbone of the loop being studied, followed by either minimization or molecular dynamics followed by minimization starting from these random structures. The details of the algorithm for the generation of the loops are presented in the first paper in this series (Shenkin et al. [submitted]). The results of minimization and molecular dynamics applied to these loops is presented here. For the two shortest CDRs studied (H1 and L2, which are five and seven amino acids long), minimizations and dynamics simulations which ignore interactions of the loop amino acids beyond the carbon beta replicate the conformation of the crystal structure closely. This suggests that these loops fold independently of sequence variation. For the third CDR (L3, which is nine amino acids), those portions of the CDR near its base which are hydrogen bonded to framework are well replicated by our procedures, but the top of the loop shows significant conformational variability. This variability persists when side chain interactions for the MCPC603 sequence are included. For a fourth CDR (H3, which is 11 amino acids long), new low-energy backbone conformations are found; however, only those which are close to the crystal are compatible with the sequence when side chain interactions are taken into account. Results from minimization and dynamics on single CDRs with all other CDRs removed are presented. These allow us to explore the extent to which individual CDR conformations are determined by interactions with framework only.     

Some thermodynamic implications for the thermostability of proteins

An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water-soluble proteins surveyed by Robertson and Murphy occurs at T* approximately 283K, independent of the heat denaturation temperature, T(m). This observation indicates, at least for these proteins, that thermostability tends to be achieved through elevation of the stability curve rather than by broadening or through a horizontal shift to higher temperatures. The relationship between the free energy of maximal stability and the temperature of heat denaturation is such that an increase in maximal stability of approximately 0.008 kJ/mole/residue is, on average, associated with a 1 degrees C increase in T(m). An estimate of the energetic consequences of thermal expansion suggests that these effects may contribute significantly to the destabilization of the native state of proteins with increasing temperature.     

Does conformational free energy distinguish loop conformations in proteins?

Limitations in protein homology modeling often arise from the inability to adequately model loops. In this paper we focus on the selection of loop conformations. We present a complete computational treatment that allows the screening of loop conformations to identify those that best fit a molecular model. The stability of a loop in a protein is evaluated via computations of conformational free energies in solution, i.e., the free energy difference between the reference structure and the modeled one. A thermodynamic cycle is used for calculation of the conformational free energy, in which the total free energy of the reference state (i.e., gas phase) is the CHARMm potential energy. The electrostatic contribution of the solvation free energy is obtained from solving the finite-difference Poisson-Boltzmann equation. The nonpolar contribution is based on a surface area-based expression. We applied this computational scheme to a simple but well-characterized system, the antibody hypervariable loop (complementarity-determining region, CDR). Instead of creating loop conformations, we generated a database of loops extracted from high-resolution crystal structures of proteins, which display geometrical similarities with antibody CDRs. We inserted loops from our database into a framework of an antibody; then we calculated the conformational free energies of each loop. Results show that we successfully identified loops with a "reference-like" CDR geometry, with the lowest conformational free energy in gas phase only. Surprisingly, the solvation energy term plays a confusing role, sometimes discriminating "reference-like" CDR geometry and many times allowing "non-reference-like" conformations to have the lowest conformational free energies (for short loops). Most "reference-like" loop conformations are separated from others by a gap in the gas phase conformational free energy scale. Naturally, loops from antibody molecules are found to be the best models for long CDRs (> or = 6 residues), mainly because of a better packing of backbone atoms into the framework of the antibody model.     

Quasirandom distribution of α and β regions in globular proteins

Distributions of α and β regions in globular proteins among clusters containing different numbers of adjacent α-helices, adjacent β regions, and “overlapping” βαβ units are considered. It is shown that these distributions do not differ greatly from what can be expected for random distributions of α and β regions along protein chains. In particular, it is shown that the amounts of relatively long α, β and βαβ clusters (which provide the basis for the conventional classification of globular proteins or domains into α, β, or α/β types) in random sequences also do not differ very much from those in real globular proteins. It follows that the possibility of structural classification of globular proteins (domains) does not imply the existence of a correlation in protein primary structure. This possibility exists even in random sequences of amino acid residues and therefore may not be the result of biological evolution.     

Detection of disordered regions in globular proteins using 13C‐detected NMR

Characterization of disordered regions in globular proteins constitutes a significant challenge. Here, we report an approach based on ¹³C‐detected nuclear magnetic resonance experiments for the identification and assignment of disordered regions in large proteins. Using this method, we demonstrate that disordered fragments can be accurately identified in two homologs of menin, a globular protein with a molecular weight over 50 kDa. Our work provides an efficient way to characterize disordered fragments in globular proteins for structural biology applications.     

Protein sequences yield a proteomic code

Analysis of crystallized protein structures suggests that globular proteins are organized as consecutively connected units of 25-35 residues. These units are closed loops, that is returns of the polypeptide chain trajectory to a close contact with itself. This universal feature of apparently polymer-statistical nature is a basis for a principally novel view on the globular proteins as loop fold structures. The same unit size has been detected in protein sequences translated from complete prokaryotic genomes by positional autocorrelation analysis, which strongly indicates the evolutionary connection of the units. The units are further characterized by prototype sequences matching to their numerous derivatives in the translated genomes. The matches to five strongest prokaryotic prototypes and three prototypes of C. elegans are identified in the sequences of crystallized proteins, and their structures analyzed. Corresponding segments of the polypeptide chains in majority of cases form closed loops, though evolutionary fate of every prototype element is shown to be rather diverse. Then loop ends can be separated by a sequence-wise distant segments and stabilized by the spatial interactions in the context of the overall globular structure. The units belong to a presumably limited spectrum of the sequence prototypes, full repertoire of which would constitute a proteomic code.     

Globular protein stability: aspects of interest in protein turnover

The conformational stability of globular proteins is remarkably low. Under physiological conditions, the native globular conformation is only from 5 to 15 kcal/mole more stable than unfolded conformations. In addition, small changes in the structure of a protein such as removing one terminal residue or cleaving a single peptide bond frequently lead to a substantial decrease in the stability. Likewise, single substitutions in the amino acid sequence can increase or decrease the stability by several kilocalories per mole. The low conformational stability of globular proteins and the sensitivity to small changes in structure suggest a possible role for conformational stability in the intracellular degradation of proteins. Several lines of evidence from in vivo studies of protein degradation are consistent with this idea.     

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.     

Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain

Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.     

Evaluation of weak interactions of proteins and organic cations with DNA duplex structures

Molecular interactions and reactions in living cells occur with high background concentrations of organic compounds including proteins. Uncharged water-soluble polymers are commonly used cosolutes in studies on molecular crowding, and most studies argue about the effects of intracellular crowding based on results obtained using polymer cosolutes. Further investigations using protein crowders and organic cations are important in understanding the effects of cellular environments on nucleic acids with negatively charged surfaces. We assessed the effects of using model globular proteins, serum proteins, histone proteins, structurally flexible polypeptides, di- and polyamines, and uncharged polymers. Thermal stability analysis of DNA oligonucleotide structures revealed that unlike conventional polymer cosolutes, basic globular proteins (lysozyme and cytochrome c) at high concentrations stabilized long internal and bulge loop structures but not fully matched duplexes. The selective stabilization of long loop structures suggests preferential binding to unpaired nucleotides in loops through weak electrostatic interactions. Furthermore, the ability of the proteins to stabilize the loop structures was enhanced under macromolecular crowding conditions. Remarkably, the effects of basic proteins on the stability of fully matched duplexes were dissimilar to those of basic amino-acid-rich polypeptides and polyamines. This study provides new insights into the interaction of nucleic acid structures with organic cations.     

Prediction of Lipid-Binding Regions in Cytoplasmic and Extracellular Loops of Membrane Proteins as Exemplified by Protein Translocation Membrane Proteins

The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation.     

Structural consensus among antibodies defines the antigen binding site

The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.