Multidomain Protein Structures Research Articles

Protein family structure signature for multidomain proteins

The rapid increase in available protein structure datasets requires new techniques for fast, yet, effective analysis of protein 3D structures. In this work, we propose a structure-based signature for protein families, suitable for rapid analysis of multidomain protein structures. Our method is alignment-free, using protein strings as the basic representation. A key novelty is the two-stage approach, whereby an initial list of candidate protein superfamilies are rapidly identified using the protein family signature, and then information retrieval methods are applied only to the members of the candidate superfamilies. This approach is the key to both improved speed, and improved structure retrieval accuracy. Experimental results, including comparative results with state-of-the-art methods, demonstrate the performance of the proposed protein family signature on queries with multidomain protein structures.

Harnessing the Combined Power of SAXS and NMR.

Single types of methodologies are no longer sufficient to adequately describe complex biological structures. As a result, integrated approaches that combine complementary data are being developed. This chapter describes the integration of nuclear magnetic resonance and small-angle scattering approaches to characterize solution structures of multi-domain proteins.

Protocol for analyzing protein ensemble structures from chemical cross-links using DynaXL

Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information. We have thus established a method called DynaXL, which allow us to extract the information from the over-length cross-links and to visualize protein ensemble structures. In this protocol, we present the detailed procedure for using DynaXL, which comprises five steps. They are identification of highly confident cross-links, delineation of protein domains/subunits, ensemble rigid-body refinement, and final validation/assessment. The DynaXL method is generally applicable for analyzing the ensemble structures of multi-domain proteins and protein–protein complexes, and is freely available at www.tanglab.org/resources.

Effect of Phosphorylation on Supertertiary Structure and Ligand Binding Affinity on Scaffold Proteins

PSD-95 and PSD-93 are multidomain scaffold proteins with a similar tertiary structure and a common set of interaction partners. Despite the high sequence homology in their protein binding domains these isoforms play opposing roles in learning and memory. The origin of their functional differences remains a mystery. The most apparent physical difference between them is the length and composition of their interdomain linkers as well as the number and position of the phosphorylation sites. In PSD-95, the binding domains partition into two structurally-independently supramodules separated by an intrinsically disordered linker. PSD-93 has a similar intrinsically disordered region but the supertertiary structure has yet to be determined. We used single molecule fluorescence resonance energy transfer (FRET) to compare the supertertiary structures and found PSD-93 has a greater separation between supramodules. This could arise from the increased linker length but also a more even distribution of positive and negative charges in PSD-93. The number and spatial organization of charges determines the extension of intrinsically-disordered peptides. Interestingly, in vitro phosphorylation by Src kinase decreased the separation in PSD-93 but increased the separation in PSD-95 resulting a similar separation between supramodules upon phosphorylation. As with structural studies, binding between PSD-95 and its ligands has been extensively studied while the affinity of PSD-93 for their shared ligands is has not been measured. Using smFRET we found that the two scaffolds have similar binding affinity, before and after phosphorylation, for their common ligands. Furthermore, we found that phosphorylation selectively increased the affinity for some ligands while leaving others unchanged. Our results show that phosphorylation can remodel the supertertiary structure of multidomain scaffold proteins. Additionally, phosphorylation can change the specificity by selectively altering binding affinity for specific ligands.

Modeling Protein Excited-state Structures from “Over-length” Chemical Cross-links

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function.

SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides for in vitro applications.

Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues.

Ensemble structure description of Lys63-linked diubiquitin.

The data described herein are related to the article entitled “Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition” [1], and to the coordinates for the ensemble structure of Lys63-linked diubiquitin (PDB code 2N2K). A Lys63-linked diubiquitin exists in three conformational states with different orientations for the two subunits, each responsible for binding to a target protein and encoding a specific cell signal. An atomic entry in the ensemble structure file consists multiple lines, representing alternative locations of the atom and recapitulating the dynamics of the protein. Experimental details about obtaining strictly intramolecular paramagnetic restraints and determining the relative occupancies of the conformational states are presented. The experimental design and procedures in this Data article can be useful for characterizing the structure and dynamics of other multi-domain proteins.

CIUSuite: A Quantitative Analysis Package for Collision Induced Unfolding Measurements of Gas-Phase Protein Ions.

Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase. CIU has been used for a variety of applications, from differentiating binding modes of activation state-selective kinase inhibitors to characterizing the domain structure of multidomain proteins. With the growing utilization of CIU as a tool for structural biology, significant challenges have emerged in data analysis and interpretation, specifically the normalization and comparison of CIU data sets. Here, we present CIUSuite, a suite of software modules designed for the rapid processing, analysis, comparison, and classification of CIU data. We demonstrate these tools as part of a series of workflows for applications in comparative structural biology, biotherapeutic analysis, and high throughput screening of kinase inhibitors. These examples illustrate both the potential for CIU in general protein analysis as well as a demonstration of best practices in the interpretation of CIU data.

Modeling Macromolecular Motions by X-Ray-Scattering-Constrained Molecular Dynamics

A fundamental problem for structural biology in combining solution x-ray scattering data with atomic resolution structures determined by crystallography is to find effective ways to model motions accessible to molecules outside the crystal environment. Motions can be derived when multiple conformations are experimentally observed or can be inferred through computational methods such as normal modes, molecular dynamics, and Monte Carlo-based techniques. Small-angle x-ray scattering (SAXS) is a complete, resolution-limited structural measurement of the solution state for macromolecules.

AIDA: ab initio domain assembly for automated multi-domain protein structure prediction and domain-domain interaction prediction.

Most proteins consist of multiple domains, independent structural and evolutionary units that are often reshuffled in genomic rearrangements to form new protein architectures. Template-based modeling methods can often detect homologous templates for individual domains, but templates that could be used to model the entire query protein are often not available. We have developed a fast docking algorithm ab initio domain assembly (AIDA) for assembling multi-domain protein structures, guided by the ab initio folding potential. This approach can be extended to discontinuous domains (i.e. domains with 'inserted' domains). When tested on experimentally solved structures of multi-domain proteins, the relative domain positions were accurately found among top 5000 models in 86% of cases. AIDA server can use domain assignments provided by the user or predict them from the provided sequence. The latter approach is particularly useful for automated protein structure prediction servers. The blind test consisting of 95 CASP10 targets shows that domain boundaries could be successfully determined for 97% of targets. The AIDA package as well as the benchmark sets used here are available for download at http://ffas.burnham.org/AIDA/. adam@sanfordburnham.org Supplementary data are available at Bioinformatics online.

Landscape of Intertwined Associations in Multi-Domain Homo-Oligomeric Proteins

This study charts the landscape of multi-domain protein structures that form intertwined homodimers by exchanging structural domains between subunits. A representative dataset of such homodimers was derived from the Protein Data Bank, and their structural and topological properties were compared to those of a representative set of non-intertwined homodimers. Most of the intertwined dimers form closed assemblies with head-to-tail arrangements, where the subunit interface involves contacts between dissimilar domains. In contrast, the non-intertwined dimers form preferentially head-to-head arrangements, where the subunit interface involves contacts between identical domains. Most of these contacts engage only one structural domain from each subunit, leaving the remaining domains free to form other associations. Remarkably, we find that multi-domain proteins closely related to the intertwined homodimers are significantly more likely than relatives of the non-intertwined versions to adopt alternative intramolecular domain arrangements. In ~40% of the intertwined dimers, the plasticity in domain arrangements among relatives affords maintenance of the head-to-head or head-to-tail topology and conservation of the corresponding subunit interface. This property seems to be exploited in several systems to regulate DNA binding. In ~58%, however, intramolecular domain re-arrangements are associated with changes in oligomeric states and poorly conserved interfaces among relatives. This time, the corresponding structural plasticity appears to be exploited by evolution to modulate function by switching between active and inactive states of the protein. Surprisingly, in total, only three systems were found to undergo the classical monomer to intertwined dimer conversion associated with three-dimensional domain swapping.

Structural Characterization of a Flexible Two-Domain Protein in Solution Using Small Angle X-Ray Scattering and NMR Data

Multidomain proteins in which individual domains are connected by linkers often possess inherent interdomain flexibility that significantly complicates their structural characterization in solution using either nuclear magnetic resonance (NMR) spectroscopy or small-angle X-ray scattering (SAXS) alone. Here, we report a protocol for joint refinement of flexible multidomain protein structures against NMR distance and angular restraints, residual dipolar couplings, and SAXS data. The protocol is based on the ensemble optimization method principle (Bernadó et al., 2007) and is compared with different refinement strategies for the structural characterization of the flexible two-domain protein sf3636 from Shigella flexneri 2a. The results of our refinement suggest the existence of a dominant population of configurational states in solution possessing an overall elongated shape and restricted relative twisting of the two domains.

Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized.

1P261 3つ以上のドメインからなるマルチドメインタンパク質の構造予測(22A. 生命情報科学:構造ゲノミクス,ポスター,第52回日本生物物理学会年会(2014年度))

1P261 3つ以上のドメインからなるマルチドメインタンパク質の構造予測(22A. 生命情報科学:構造ゲノミクス,ポスター,第52回日本生物物理学会年会(2014年度))

Molecular Crowding Enhanced ATPase Activity of the RNA Helicase eIF4A Correlates with Compaction of Its Quaternary Structure and Association with eIF4G

Enzymatic reactions occur in a crowded and confined environment in vivo, containing proteins, RNA and DNA. Previous reports have shown that interactions between macromolecules, and reactions rates differ significantly between crowded environments and dilute buffers. However, the direct effect of crowding on the level of high-resolution structures of macromolecules has not been extensively analyzed and is not well understood. Here we analyze the effect of macromolecular crowding on structure and function of the human translation initiation factors eIF4A, a two-domain DEAD-Box helicase, the HEAT-1 domain of eIF4G, and their complex. We find that crowding enhances the ATPase activity of eIF4A, which correlates with a shift to a more compact structure as revealed with small-angle X-ray scattering. However, the individual domains of eIF4A, or the eIF4G-HEAT-1 domain alone show little structural changes due to crowding except for flexible regions. Thus, the effect of macromolecular crowding on activity and structure need to be taken into account when evaluating enzyme activities and structures of multidomain proteins, proteins with flexible regions, or protein complexes obtained by X-ray crystallography, NMR, or other structural methods.

Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.

Evidence that the amino acid residue Ile121 is involved in arginine kinase activity and structural stability

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Domain–domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of amino acid residues located in the linker between the N- and C-terminal domains in the structural stability and functions of multidomain proteins. In this research, A series mutation of conserved residue Ile121 located in the linker were mutated to explore its roles in the activity and structural stability of AK. The mutations I121D and I121K led to pronounced loss of activity and structural stability. Furthermore, these mutations also led to serious aggregation during heat-and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, all the mutantions except I121L could not successfully recover their activities by dilution-initiated refolding, and showed significant decreased rate constant during AK refolding. While the mutation I121L almost had no effect on AK activity and structural stability. These results suggested that mutations of the residue I121 in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the N- and C-terminal domains.

DINE: A Novel Score Function for Modeling Multidomain Protein Structures with Domain Linker and Interface Restraints

DINE: A Novel Score Function for Modeling Multidomain Protein Structures with Domain Linker and Interface Restraints

DINE: A Novel Score Function for Modeling Multidomain Protein Structures with Domain Linker and Interface Restraints

DINE: A Novel Score Function for Modeling Multidomain Protein Structures with Domain Linker and Interface Restraints