Designing proteins to crystallize through beta-strand pairing

Inherent difficulties in growing protein crystals are major concerns within structural biology and particularly in structural proteomics. Here, we describe a novel approach of engineering target proteins by surface mutagenesis to increase the odds of crystallizing the molecules. To this end, we have exploited our recent triad-hypothesis using proteins with crystallographically defined beta-structures as the principal models. Crystal packing analyses of 182 protein structures belonging to 21 different superfamilies implied that the propensities to crystallize could be engineered into target proteins by replacing short segments, 5-6 residues, of their beta-strands with 'cassettes' of suitable packing motifs. These packing motifs will generate specific crystal packing interactions that promote crystallization. Key features of the primary and tertiary structures of such packing motifs have been identified for immunoglobulins. Further, packing motifs have been engineered successfully into six model antibodies without disturbing their capabilities to be produced, their immunoreactivity and their overall structure. Preliminary crystallization analyses have also been performed. Taken together, the procedures outline a rational protocol for crystallizing proteins by surface mutagenesis. The importance of these findings is discussed in relation to the crystallization of proteins in general.     

Secondary structure based analysis and classification of biological interfaces: identification of binding motifs in protein-protein interactions

MOTIVATION: The increasing amount of data on protein-protein interaction needs to be rationalized for deriving guidelines for the alteration or design of an interface between two proteins. RESULTS: We present a detaild structural analysis and comparison of homo- versus heterodimeric protein-protein interfaces. Regular secondary structures (helices and strands) are the main components of the former, whereas non-regular structures (turns, loops, etc.) frequently mediate interactions in the latter. Interface helices get longer with increasing interface area, but only in heterocomplexes. On average, the homodimers have longer helical segments and prominent helix-helix pairs. There is a surprising distinction in the relative orientation of interface helices, with a tendency for aligned packing in homodimers and a clear preference for packing at 90 degrees in heterodimers. Arg and the aromatic residues have a higher preference to occur in all secondary structural elements (SSEs) in the interface. Based on the dominant SSE, the interfaces have been grouped into four classes: alpha, beta, alphabeta and non-regular. Identity between protein and interface classes is the maximum for alpha proteins, but rather mediocre for the other protein classes. The interface classes of the two chains forming a heterodimer are often dissimilar. Eleven binding motifs can capture the prominent architectural features of most of the interfaces.     

Decomposing the space of protein quaternary structures with the interface fragment pair library

Background

The physical interactions between proteins constitute the basis of protein quaternary structures. They dominate many biological processes in living cells. Deciphering the structural features of interacting proteins is essential to understand their cellular functions. Similar to the space of protein tertiary structures in which discrete patterns are clearly observed on fold or sub-fold motif levels, it has been found that the space of protein quaternary structures is highly degenerate due to the packing of compact secondary structure elements at interfaces. Therefore, it is necessary to further decompose the protein quaternary structural space into a more local representation.

Results

Here we constructed an interface fragment pair library from the current structure database of protein complexes. After structural-based clustering, we found that more than 90% of these interface fragment pairs can be represented by a limited number of highly abundant motifs. These motifs were further used to guide complex assembly. A large-scale benchmark test shows that the native-like binding is highly likely in the structural ensemble of modeled protein complexes that were built through the library.

Conclusions

Our study therefore presents supportive evidences that the space of protein quaternary structures can be represented by the combination of a small set of secondary-structure-based packing at binding interfaces. Finally, after future improvements such as adding sequence profiles, we expect this new library will be useful to predict structures of unknown protein-protein interactions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0437-4) contains supplementary material, which is available to authorized users.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

Sequence motifs,polar interactions and conformational changes in helical membrane proteins

The alpha helices of transmembrane proteins interact to form higher order structures. These interactions are frequently mediated by packing motifs (such as GxxxG) and polar residues. Recent structural data have revealed that small sidechains are able to both stabilize helical membrane proteins and allow conformational changes in the structure. The strong interactions involving polar sidechains often contribute to protein misfolding or malfunction.     

Protein-protein crystal-packing contacts.

Protein-protein contacts in monomeric protein crystal structures have been analyzed and compared to the physiological protein-protein contacts in oligomerization. A number of features differentiate the crystal-packing contacts from the natural contacts occurring in multimeric proteins. The area of the protein surface patches involved in packing contacts is generally smaller and its amino acid composition is indistinguishable from that of the protein surface accessible to the solvent. The fraction of protein surface in crystal contacts is very variable and independent of the number of packing contacts. The thermal motion at the crystal packing interface and that of the protein core, even for large packing interfaces, though the tendency is to be closer to that of the core. These results suggest that protein crystallization depends on random protein-protein interactions, which have little in common with physiological protein-protein recognition processes, and that the possibility of engineering macromolecular crystallization to improve crystal quality could be widened.     

Protein domain interfaces: characterization and comparison with oligomeric protein interfaces

The physical and chemical properties of domain-domain interactions have been analysed in two-domain proteins selected from the protein classification, CATH. The two-domain structures were divided into those derived from (i) monomeric proteins, or (ii) oligomeric or complexed proteins. The size, polarity, hydrogen bonding and packing of the intra-chain domain interface were calculated for both sets of two-domain structures. The results were compared with inter-chain interface parameters from permanent and non-obligate protein-protein complexes. In general, the intra-chain domain and inter-chain interfaces were remarkably similar. Many of the intra-chain interface properties are intermediate between those calculated for permanent and non-obligate inter-chain complexes. Residue interface propensities were also found to be very similar, with hydrophobic residues playing a major role, together with positively charged arginine residues. In addition, the residue composition of the domain interfaces were found to be more comparable with domain surfaces than domain cores. The implications of these results for domain swapping and protein folding are discussed.     

A dissection of specific and non-specific protein-protein interfaces

We compare the geometric and physical-chemical properties of interfaces involved in specific and non-specific protein-protein interactions in crystal structures reported in the Protein Data Bank. Specific interactions are illustrated by 70 protein-protein complexes and by subunit contacts in 122 homodimeric proteins; non-specific interactions are illustrated by 188 pairs of monomeric proteins making crystal-packing contacts selected to bury more than 800 A2 of protein surface. A majority of these pairs have 2-fold symmetry and form "crystal dimers" that cannot be distinguished from real dimers on the basis of the interface size or symmetry. The chemical and amino acid compositions of the large crystal-packing interfaces resemble the protein solvent-accessible surface. These interfaces are less hydrophobic than in homodimers and contain much fewer fully buried atoms. We develop a residue propensity score and a hydrophobic interaction score to assess preferences seen in the chemical and amino acid compositions of the different types of interfaces, and we derive indexes to evaluate the atomic packing, which we find to be less compact at non-specific than at specific interfaces. We test the capacity of these parameters to identify homodimeric proteins in crystal structures, and show that a simple combination of the non-polar interface area and the fraction of buried interface atoms assigns the quaternary structure of 88% of the homodimers and 77% of the monomers in our data set correctly. These success rates increase to 93-95% when the residue propensity score of the interfaces is taken into consideration.     

Peptide segments in protein-protein interfaces

An important component of functional genomics involves the understanding of protein association. The interfaces resulting from protein-protein interactions - (i) specific, as represented by the homodimeric quaternary structures and the complexes formed by two independently occurring protein components, and (ii) non-specific, as observed in the crystal lattice of monomeric proteins - have been analysed on the basis of the length and the number of peptide segments. In 1000 A2 of the interface area, contributed by a polypeptide chain, there would be 3.4 segments in homodimers, 5.6 in complexes and 6.3 in crystal contacts. Concomitantly, the segments are the longest (with 8.7 interface residues) in homodimers. Core segments (likely to contribute more towards binding) are more in number in homodimers (1.7) than in crystal contacts (0.5), and this number can be used as one of the parameters to distinguish between the two types of interfaces. Dominant segments involved in specific interactions, along with their secondary structural features, are enumerated.     

Geometric Similarities of Protein-Protein Interfaces at Atomic Resolution Are Only Observed within Homologous Families: An Exhaustive Structural Classification Study

To elucidate the structural basis of the diversity and universality in protein-protein interactions, an exhaustive all-against-all structural comparison of all known protein interfaces in the Protein Data Bank was performed at atomic resolution. After similar interfaces were clustered, approximately 20,000 structural motifs with at least two members were identified, out of which 3678 motifs consisted of at least 10 interfaces. Except for some trivial interfaces involving single α helices, almost all motifs were found to be confined within single protein families. Furthermore, the interaction partners of each motif were found to be very limited, and, accordingly, the interaction networks of the motifs tend to be small and are much more restricted than the binding sites for small ligand molecules. These findings suggest that, at the level of atomic structures, protein-protein interactions are precisely designed; hence, protein interfaces with multiple interacting partners should involve incompletely overlapping multiple interfaces and/or accommodate structural changes upon binding to their targets.     

Structural aspects of recognition motifs contributing to autoimmune responses.

Sequence analysis of autoimmune-associated antibodies has suggested a structural relatedness between genes used to encode autoantibodies and those encoding unrelated antibodies without autoreactive specificities. Subsequently, the basis for cross-reactive idiotypes across germ-line lineages, as well as conserved interspecies cross-reactivities of autoantigens among serologically similar antibodies, may result from evolutionary duplication of particular types of recognition motifs. As a first step toward elucidating structural recognition principles underlying possible cross-reactive epitopes involved in autoimmune pathologies, structural features of selected motifs associated with native ligand binding are examined for their inherent occurrence in antibody and T-cell receptor repertoires. This analysis considers the putative recognition features representative of common motif subsets shared with loop structures in CDR2 and FR3 regions of antibodies such as charge-2x-charge-x-charge or hydrogen bond donor (acceptor)-2x-charge-x-hydrogen bond donor (acceptor) type motifs, where x is any residue that can participate in maintaining a loop conformation. Such tracts encoded in the CRD2 and FR3 regions of heavy chains of antibodies and T-cell receptors (TCRs) associated with autoimmune dysfunction, with non-autoreactive antibodies, and with native host proteins. Such evolutionarily conserved motifs may be targets for complementary interactions involving autoantibodies and receptors.     

Thermodynamic Characterization of Conformational States of Biological Macromolecules using Differential Scanning Calorimetry

Abstract

The Ras superfamily small G proteins are master regulators of a diverse range of cellular processes and act via downstream effector molecules. The first structure of a small G protein–effector complex, that of Rap1A with c-Raf1, was published 20 years ago. Since then, the structures of more than 60 small G proteins in complex with their effectors have been published. These effectors utilize a diverse array of structural motifs to interact with the G protein fold, which we have divided into four structural classes: intermolecular β-sheets, helical pairs, other interactions, and pleckstrin homology (PH) domains. These classes and their representative structures are discussed and a contact analysis of the interactions is presented, which highlights the common effector-binding regions between and within the small G protein families.     

Structural Variation and Uniformity among Tetraloop-Receptor Interactions and Other Loop-Helix Interactions in RNA Crystal Structures

Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the “standard” GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.     

Structures composing protein domains

This review summarizes available data concerning intradomain structures (IS) such as functionally important amino acid residues, short linear motifs, conserved or disordered regions, peptide repeats, broadly occurring secondary structures or folds, etc. IS form structural features (units or elements) necessary for interactions with proteins or non-peptidic ligands, enzyme reactions and some structural properties of proteins. These features have often been related to a single structural level (e.g. primary structure) mostly requiring certain structural context of other levels (e.g. secondary structures or supersecondary folds) as follows also from some examples reported or demonstrated here. In addition, we deal with some functionally important dynamic properties of IS (e.g. flexibility and different forms of accessibility), and more special dynamic changes of IS during enzyme reactions and allosteric regulation. Selected notes concern also some experimental methods, still more necessary tools of bioinformatic processing and clinically interesting relationships.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Similar binding sites and different partners: implications to shared proteins in cellular pathways

We studied a data set of structurally similar interfaces that bind to proteins with different binding-site structures and different functions. Our multipartner protein interface clusters enable us to address questions like: What makes a given site bind different proteins? How similar/different are the interactions? And, what drives the apparently less-specific association? We find that proteins with common binding-site motifs preferentially use conserved interactions at similar interface locations, despite the different partners. Helices are major vehicles for binding different partners, allowing alternate ways to achieve favorable association. The binding sites are characterized by imperfect packing, planar architectures, bridging water molecules, and, on average, smaller size. Interestingly, analysis of the connectivity of these proteins illustrates that they have more interactions with other proteins. These findings are important in predicting "date hubs," if we assume that "date hubs" are shared proteins with binding sites capable of transient binding to multipartners, linking higher-order networks.     

Glycine residues G338 and G342 are important determinants for serotonin transporter dimerisation and cell surface expression

Compelling evidence has been provided that Na(+) and Cl(-)-dependent neurotransmitter transporter proteins form oligomeric complexes. Specific helix-helix interactions in lipid bilayers are thought to promote the assembly of integral membrane proteins to oligomeric structures. These interactions are determined by selective transmembrane helix packing motifs one of which is the Glycophorin A motif (GxxxG). This motif is present in the sixth transmembrane domain of most transporter proteins. In order to investigate, whether this motif is important for proper expression and function of the serotonin transporter (SERT), we have analysed the effect of mutating the respective glycine residues Gly338 and Gly342 to valine upon transient expression of the respective cDNAs in HEK293 cells. As revealed by western blotting, wildtype SERT is found in monomeric and dimeric forms while both mutants are expressed as monomers solely. Confocal microscopy revealed that the wildtype SERT is expressed at the cell surface, whereas both mutant proteins are localised in intracellular compartments. Failure of integration into the cell membrane is responsible for a total loss of [(3)H]5HT uptake capability by the mutants. These findings show that in the SERT protein the integrity of the GxxxG motif is essential for dimerisation and proper targeting of the transporter complex to the cell surface.     

Plasticity of quaternary structure: twenty-two ways to form a LacI dimer

The repressor proteins of the LacI/GalR family exhibit significant similarity in their secondary and tertiary structures despite less than 35% identity in their primary sequences. Furthermore, the core domains of these oligomeric repressors, which mediate dimerization, are homologous with the monomeric periplasmic binding proteins, extending the issue of plasticity to quaternary structure. To elucidate the determinants of assembly, a structure-based alignment has been created for three repressors and four periplasmic binding proteins. Contact maps have also been constructed for the three repressor interfaces to distinguish any conserved interactions. These analyses show few strict requirements for assembly of the core N-subdomain interface. The interfaces of repressor core C-subdomains are well conserved at the structural level, and their primary sequences differ significantly from the monomeric periplasmic binding proteins at positions equivalent to LacI 281 and 282. However, previous biochemical and phenotypic analyses indicate that LacI tolerates many mutations at 281. Mutations at LacI 282 were shown to abrogate assembly, but for Y282D this could be compensated by a second-site mutation in the core N-subdomain at K84 to L or A. Using the link between LacI assembly and function, we have further identified 22 second-site mutations that compensate the Y282D dimerization defect in vivo. The sites of these mutations fall into several structural regions, each of which may influence assembly by a different mechanism. Thus, the 360-amino acid scaffold of LacI allows plasticity of its quaternary structure. The periplasmic binding proteins may require only minimal changes to facilitate oligomerization similar to the repressor proteins.     

Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states

Actin filament growth and disassembly, as well as affinity for actin-binding proteins, is mediated by the nucleotide-bound state of the component actin monomers. The structural differences between ATP-actin and ADP-actin, however, remain controversial. We expressed a cytoplasmic actin in Sf9 cells, which was rendered non-polymerizable by virtue of two point mutations in subdomain 4 (A204E/P243K). This homogeneous monomer, called AP-actin, was crystallized in the absence of toxins, binding proteins, or chemical modification, with ATP or ADP at the active site. The two surface mutations do not perturb the structure. Significant differences between the two states are confined to the active site region and sensor loop. The active site cleft remains closed in both states. Minor structural shifts propagate from the active site toward subdomain 2, but dissipate before reaching the DNase binding loop (D-loop), which remains disordered in both the ADP and ATP states. This result contrasts with previous structures of actin made monomeric by modification with tetramethylrhodamine, which show formation of an alpha-helix at the distal end of the D-loop in the ADP-bound but not the ATP-bound form (Otterbein, L. R., Graceffa, P., and Dominguez, R. (2001) Science 293, 708-711). Our reanalysis of the TMR-modified actin structures suggests that the nucleotide-dependent formation of the D-loop helix may result from signal propagation through crystal packing interactions. Whereas the observed nucleotide-dependent changes in the structure present significantly different surfaces on the exterior of the actin monomer, current models of the actin filament lack any actin-actin interactions that involve the region of these key structural changes.