NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.

How Effective Is The Data On Co-Occurrence Of Domains In Multi-Domain Proteins In Prediction Of Protein-Protein Interactions?

We explore the fuse of information on co-occurrence of domains in multi-domain proteins in predicting protein-protein interactions. The basic premise of our work is the assumption that domains co-occurring in a polypeptide chain undergo either structural or functional interactions among themselves. In this study we use a template dataset of domains in multidomain proteins and predict protein-protein interactions in a target organism. We note that maximum number of correct predictions of interacting protein domain families (158) is made in S. cerevisiae when the dataset of closely related organisms is used as the template followed by the more diverse dataset of bacterial proteins (48) and a dataset of randomly chosen proteins (23). We conclude that use of multi-domain information from organisms closely-related to the target can aid prediction of interacting protein families.

Generation of Flat and Curved Membranes by Inverse-BAR Domain Proteins

Biological membranes are tightly linked to the evolution of life, because they provide a way to concentrate molecules into partially closed compartments. The dynamic shaping of cellular membranes is essential for many physiological processes, including cell morphogenesis, motility, cytokinesis, endocytosis, and secretion. It is therefore essential to understand the structure of the membrane and recognize the players that directly sculpt the membrane and enable it to adopt different shapes. The actin cytoskeleton provides the force to push eukaryotic plasma membrane in order to form different protrusions or/and invaginations. It has now became evident that actin directly co-operates with many membrane sculptors, including BAR domain proteins, in these important events. However, the molecular mechanisms behind BAR domain function and the differences between the members of this large protein family remain largely unresolved. In this thesis, the structure and functions of the I-BAR domain family members IRSp53 and MIM were thoroughly analyzed. By using several methods such as electron microscopy and systematic mutagenesis, we showed that these I-BAR domain proteins bind to PI(4,5)P2-rich membranes, generate negative membrane curvature and are involved in the formation of plasma membrane protrusions in cells e.g. filopodia. Importantly, we characterized a novel member of the BAR-domain superfamily which we named Pinkbar. We revealed that Pinkbar is specifically expressed in kidney and epithelial cells, and it localizes to Rab13-positive vesicles in intestinal epithelial cells. Remarkably, we learned that the I-BAR domain of Pinkbar does not generate membrane curvature but instead stabilizes planar membranes. Based on structural, mutagenesis and biochemical work we present a model for the mechanism of the novel membrane deforming activity of Pinkbar. Collectively, this work describes the mechanism by which I-BAR domain proteins deform membranes and provides new information about the biological roles of these proteins. Intriguingly, this work also gives evidence that significant functional plasticity exists within the I-BAR domain family. I-BAR proteins can either generate negative membrane curvature or stabilize planar membrane sheets, depending on the specific structural properties of their I-BAR domains. The results presented in this thesis expand our knowledge on membrane sculpting mechanisms and shows for the first time how flat membranes can be generated in cells.

Stability of domain structures in multi-domain proteins

Multi-domain proteins have many advantages with respect to stability and folding inside cells. Here we attempt to understand the intricate relationship between the domain-domain interactions and the stability of domains in isolation. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Stability of such folds to exist independently is optimized by evolution. Specific residue mutations in the sites equivalent to inter-domain interface enhance the overall solvation, thereby stabilizing these domain folds independently. A few naturally occurring variants at these sites alter communication between domains and affect stability leading to disease manifestation. Our analysis provides safe guidelines for mutagenesis which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR.

Tethering preferences of domain families co-occurring in multi-domain proteins

Genomic data of several organisms have revealed the presence of a vast repertoire of multi-domain proteins. The role played by individual domains in a multi-domain protein has a profound influence on the overall function of the protein. In the present analysis an attempt has been made to better understand the tethering preferences of domain families that occur in multi-domain proteins. The analysis has been carried out on an exhaustive dataset of 2 961 898 sequences of proteins from 930 organisms, where 741 274 proteins are comprised of at least two domain families. For every domain family, the number of other domain families with which it co-occurs within a protein in this dataset has been enumerated and is referred to as the tethering number of the domain family. It was found that, in the general dataset, the AAA ATPase family and the family of Ser/Thr kinases have the highest tethering numbers of 450 and 444 respectively. Further analysis reveals significant correlation between the number of members in a family and its tethering number. Positive correlation was also observed for the extent of a sequence and functional diversity within a family and the tethering numbers of domain families. Domain families that are present ubiquitously in diverse organisms tend to have large tethering numbers, while organism/kingdom-specific families have low tethering numbers. Thus, the analysis uncovers how domain families recombine and evolve to give rise to multi-domain proteins.

Understanding the role of domain-domain linkers in the spatial orientation of domains in multi-domain proteins

Inter-domain linkers (IDLs)' bridge flanking domains and support inter-domain communication in multi-domain proteins. Their sequence and conformational preferences enable them to carry out varied functions. They also provide sufficient flexibility to facilitate domain motions and, in conjunction with the interacting interfaces, they also regulate the inter-domain geometry (IDG). In spite of the basic intuitive understanding of the inter-domain orientations with respect to linker conformations and interfaces, we still do not entirely understand the precise relationship among the three. We show that IDG is evolutionarily well conserved and is constrained by the domain-domain interface interactions. The IDLs modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. Results of our analysis provide guidelines in modelling of multi-domain proteins from the tertiary structures of constituent domain components.

The relationship between classification of multi-domain proteins using an alignment-free approach and their functions: a case study with immunoglobulins

Establishing functional relationships between multi-domain protein sequences is a non-trivial task. Traditionally, delineating functional assignment and relationships of proteins requires domain assignments as a prerequisite. This process is sensitive to alignment quality and domain definitions. In multi-domain proteins due to multiple reasons, the quality of alignments is poor. We report the correspondence between the classification of proteins represented as full-length gene products and their functions. Our approach differs fundamentally from traditional methods in not performing the classification at the level of domains. Our method is based on an alignment free local matching scores (LMS) computation at the amino-acid sequence level followed by hierarchical clustering. As there are no gold standards for full-length protein sequence classification, we resorted to Gene Ontology and domain-architecture based similarity measures to assess our classification. The final clusters obtained using LMS show high functional and domain architectural similarities. Comparison of the current method with alignment based approaches at both domain and full-length protein showed superiority of the LMS scores. Using this method we have recreated objective relationships among different protein kinase sub-families and also classified immunoglobulin containing proteins where sub-family definitions do not exist currently. This method can be applied to any set of protein sequences and hence will be instrumental in analysis of large numbers of full-length protein sequences.

Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: Implications for three-dimensional modeling

With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naive Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (approximate to 85%) and specific (approximate to 95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. Proteins 2014; 82:1219-1234. (c) 2013 Wiley Periodicals, Inc.

CLAP: A web-server for automatic classification of proteins with special reference to multi-domain proteins

Background: The function of a protein can be deciphered with higher accuracy from its structure than from its amino acid sequence. Due to the huge gap in the available protein sequence and structural space, tools that can generate functionally homogeneous clusters using only the sequence information, hold great importance. For this, traditional alignment-based tools work well in most cases and clustering is performed on the basis of sequence similarity. But, in the case of multi-domain proteins, the alignment quality might be poor due to varied lengths of the proteins, domain shuffling or circular permutations. Multi-domain proteins are ubiquitous in nature, hence alignment-free tools, which overcome the shortcomings of alignment-based protein comparison methods, are required. Further, existing tools classify proteins using only domain-level information and hence miss out on the information encoded in the tethered regions or accessory domains. Our method, on the other hand, takes into account the full-length sequence of a protein, consolidating the complete sequence information to understand a given protein better. Results: Our web-server, CLAP (Classification of Proteins), is one such alignment-free software for automatic classification of protein sequences. It utilizes a pattern-matching algorithm that assigns local matching scores (LMS) to residues that are a part of the matched patterns between two sequences being compared. CLAP works on full-length sequences and does not require prior domain definitions. Pilot studies undertaken previously on protein kinases and immunoglobulins have shown that CLAP yields clusters, which have high functional and domain architectural similarity. Moreover, parsing at a statistically determined cut-off resulted in clusters that corroborated with the sub-family level classification of that particular domain family. Conclusions: CLAP is a useful protein-clustering tool, independent of domain assignment, domain order, sequence length and domain diversity. Our method can be used for any set of protein sequences, yielding functionally relevant clusters with high domain architectural homogeneity. The CLAP web server is freely available for academic use at http://nslab.mbu.iisc.ernet.in/clap/.

Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris

RpfG is a paradigm for a class of widespread bacterial two-component regulators with a CheY-like receiver domain attached to a histidine-aspartic acid-glycine-tyrosine-proline (HD-GYP) cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), a two-component system comprising RpfG and the complex sensor kinase RpfC is implicated in sensing and responding to the diffusible signaling factor (DSF), which is essential for cell-cell signaling. RpfF is involved in synthesizing DSF, and mutations of rpfF, rpfG, or rpfC lead to a coordinate reduction in the synthesis of virulence factors such as extracellular enzymes, biofilm structure, and motility. Using yeast two-hybrid analysis and fluorescence resonance energy transfer experiments in Xcc, we show that the physical interaction of RpfG with two proteins with diguanylate cyclase (GGDEF) domains controls a subset of RpfG-regulated virulence functions. RpfG interactions were abolished by alanine substitutions of the three residues of the conserved GYP motif in the HD-GYP domain. Changing the GYP motif or deletion of the two GGDEF-domain proteins reduced Xcc motility but not the synthesis of extracellular enzymes or biofilm formation. RpfG-GGDEF interactions are dynamic and depend on DSF signaling, being reduced in the rpfF mutant but restored by DSF addition. The results are consistent with a model in which DSF signal transduction controlling motility depends on a highly regulated, dynamic interaction of proteins that influence the localized expression of cyclic di-GMP.

Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris, RpfG together with the sensor kinase RpfC regulates multiple factors as a response to the cell-to-cell Diffusible Signalling Factor (DSF). A dynamic physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. Here we show that, contrary to expectation, regulation of motility by the GGDEF domain proteins does not depend upon their cyclic di-GMP synthetic activity. Furthermore we show that the complex of RpfG and GGDEF domain proteins recruits a specific PilZ domain adaptor protein, and this complex then interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signalling influences motility through the highly regulated dynamic interaction of proteins that affect pilus action. A specific motif that we identify to be required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions is of broad relevance.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

Stretching single-domain proteins: Phase diagram and kinetics of force-induced unfolding

Single-molecule force spectroscopy reveals unfolding of domains in titin on stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single-domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively, whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a stepwise “quantized” manner. Unfolding dynamics and forces required to stretch proteins depend sensitively on topology. The unfolding rates increase exponentially with force f till an optimum value, which is determined by the barrier to unfolding when f = 0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single-molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants.

Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN

The four mammalian golgins, p230/golgin-245, golgin-97, GCC88 and GCC185 are targeted to trans-Golgi network ITGN) membranes by their C-terminal GRIP domain in a G-protein-dependent process. The Arf-like GTPase, Arl1, has been shown to mediate TGN recruitment of p230/golgin245 and golgin-97 by interaction with their GRIP domains; however, it is not known whether all the TGN golgins bind to Arl1 and whether they are all recruited to the same or different TGN domains. Here we demonstrate differences in membrane binding properties and TGN domain recruitment of the mammalian GRIP domain proteins. Overexpression of full-length GCC185 resulted in the appearance of small punctate structures dispersed in the cytoplasm of transfected cells that were identified as membrane tubular structures by immunoelectron microscopy. The cytoplasmic GCC185-labelled structures were enriched for membrane binding determinants of GCC185 GRIP, whereas the three other mammalian GRIP family members did not colocalize with the GCC185-labelled structures. These GCC185-labelled structures included the TGN resident protein alpha2,6 sialyltransferase and excluded the recycling TGN protein, TGN46. The Golgi stack was unaffected by overexpression of GCC185. Overexpression of both full-length GCC185 and GCC88 showed distinct and nonoverlapping structures. We also show that the GRIP domains of GCC185 and GCC88 differ in membrane binding properties from each other and, in contrast to p230/golgin245 and golgin-97, do not interact with Arl1 in vivo. Collectively these results show that GCC88, GCC185 and p230/golgin245 are recruited to functionally distinct domains of the TGN and are likely to be important for the maintenance of TGN subdomain structure, a critical feature for mediating protein sorting and membrane transport.