Getting a grip on complexes

Abstract

We are witnessing tremendous advances in our understanding of the organization of life. Complete genomes are being deciphered with ever increasing speed and accuracy, thereby setting the stage for addressing the entire gene product repertoire of cells, towards understanding whole biological systems. Advances in bioinformatics and mass spectrometric techniques have revealed the multitude of interactions present in the proteome. Multiprotein complexes are emerging as a paramount cornerstone of biological activity, as many proteins appear to participate, stably or transiently, in large multisubunit assemblies. Analysis of the architecture of these assemblies and their manifold interactions is imperative for understanding their function at the molecular level. Structural genomics efforts have fostered the development of many technologies towards achieving the throughput required for studying system-wide single proteins and small interaction motifs at high resolution. The present shift in focus towards large multiprotein complexes, in particular in eukaryotes, now calls for a likewise concerted effort to develop and provide new technologies that are urgently required to produce in quality and quantity the plethora of multiprotein assemblies that form the complexome, and to routinely study their structure and function at the molecular level. Current efforts towards this objective are summarized and reviewed in this contribution.

Keywords: ACEMBL; BEVS; Proteome; complexomics.; interactome; multiBac; multigene expression; multiprotein assemblies; robotics; structural genomics.

Fig. (1)

Interactomics. Recent technological advances in genome-wide methods enable researchers to address protein-protein interactions present in the proteome of organisms in a comprehensive fashion, thus giving rise to the interactome. Native purification of proteins present in organelles and entire cells by using tandem affinity purification (TAP) methods, Strep-protein interaction experiment (SPINE) and transgenomics involving bacterial artificial chromosomes for generating stable mammalian cell lines, as well as protein-protein screens by yeast two-hybrid (Y2H) methods are supported by bioinformatics analyses, and together provide a (growing) picture of the interactome as a complex mixture of multiprotein assemblies. Mass spectrometry (MS) based proteomic methods including matrix-assisted laser desorption ionization (MALDI) and electro-spray ionization (ESI) techniques coupled to liquid chromatography (LC-MS) and tandem MS-MS measurements add to the catalogue of tools employed to tackle the complexome. The link between ineractome research and structural biology is made by native mass spectrometry. Native MS can provide vital information about the structure, topology and architecture of protein complexes preserved in the gaseous phase. Ion mobility separation coupled to mass spectrometry (IM-MS) and collision induced dissociation (CID) are new approaches holding particular promise for characterizing the properties and composition of even very large protein complexes. Recombinant overproduction, functional characterization and eventually 3-D structure determination can help to validate the vast amounts of interactome data from recent systems biology efforts. Multiplexed and quantitative MS methods in conjunction with limited proteolysis may become critically important to elucidate variants of recombinantly overproduced multiprotein complexes amenable to high-resolution structural and functional analysis. Combinatorial multigene generation, parallel small-scale expression and biochemical and biophysical analysis of multiprotein complexes derived from interactome data constitute likely modules of a conceptual “complexomics“ pipeline in analogy to current structural genomics approaches, leading to routine and rapid elucidation of the molecular architecture of many complexes and their subunit components by X-ray diffraction analysis, electron microscopy and NMR spectroscopy.

Fig. (2)

MultiBac BEVS: Eukaryotic multiprotein expression. ORFs (a-e) encoding for subunits of a protein complex and auxiliary protein such as modifiers or chaperones, are inserted into a plasmid containing the sequences required for Tn7 transposition (Tn7L, Tn7R), or a plasmid containing a LoxP imperfect inverted repeat, respectively. Gene insertion occurs via a multiplication module (small rectangles) designed for facilitating multigene cassette generation. A baculovirus genome containing the Tn7 attachment site (attn7) and a LoxP sequence, in addition to deletions beneficial for protein production, is present in bacterial cells in form of a bacterial artificial chromosome (BAC). Integration of multigene expression cassettes is mediated by the Tn7 transposon and Cre recombinase, respectively, which are expressed from helper vectors in the bacteria [73]. Transfection of insect cells with the resulting composite baculovirus results in high-level expression of the proteins in cultured insect cells. Adapted from [95].

Fig. (3)

ACEMBL System. ACEMBL consists of newly designed, small vectors (A) and automated procedures and routines relying on recombineering for gene insertion and vector fusion (B). Multigene expression constructs are generated by insertion of genes into multiple integration elements (MIE) by recombination, followed by Cre-LoxP fusion of Donors with an Acceptor. Incubation of educt constructs (here pDK, pDS, pACE) containing genes of interest (white arrows) results in all possible combinations in a single reaction including Acceptor-Donor (AD) and Acceptor-Donor-Donor (ADD) fusions as shown here schematically. Creation of even four-plasmid ADDD constructs has also been completed successfully in our laboratory [82]. All co-existing constructs have characteristic antibiotic marker combinations and resistance levels (right). Donor vectors contain a conditional origin of replication derived from R6Kγ, and thus act as suicide vectors in cloning strains devoid of the pir gene unless fused to an Acceptor with a regular replicon. A second Acceptor, pACE2, is identical to pACE except for the encoded marker which confers resistance to tetracycline rather than ampicillin (not shown). Plasmid pACE2 can be used in conjunction with pACE derivatives for example to co-express auxiliary proteins such as chaperones or modifiers [82]. (C) Recombineering workflow by using the ACEMBL system is shown. Genes are integrated in Donors or Acceptors by ligation independent methods such as SLIC followed by combinatorial multigene vector generation using Cre-LoxP fusion. Expression and purification provide protein complex for analysis. Multigene vectors are deconstructed by using Cre excision activity (De-Cre). Encoded genes are modified by PCR and reintegrated into the workflow by recombination in an iterative cycle. The entire process is compatible with automation, and was successfully scripted into a robotic routine. Adapted in part from [82, 83].