A Fifth of the Protein World: Rossmann-like Proteins as an Evolutionarily Successful Structural unit

The Rossmann-like fold is the most prevalent and diversified doubly-wound superfold of ancient evolutionary origin. Rossmann-like domains are present in a variety of metabolic enzymes and are capable of binding diverse ligands. Discerning evolutionary relationships among these domains is challenging because of their diverse functions and ancient origin. We defined a minimal Rossmann-like structural motif (RLM), identified RLM-containing domains among known 3D structures (20%) and classified them according to their homologous relationships. New classifications were incorporated into our Evolutionary Classification of protein Domains (ECOD) database. We defined 156 homology groups (H-groups), which were further clustered into 123 possible homology groups (X-groups). Our analysis revealed that RLM-containing proteins constitute approximately 15% of the human proteome. We found that disease-causing mutations are more frequent within RLM domains than within non-RLM domains of these proteins, highlighting the importance of RLM-containing proteins for human health.     

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.     

Connexin45 Interacts with Zonula Occludens-1 in Osteoblastic Cells

Connexin43(Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.     

Structural order in Pannexin 1 cytoplasmic domains

Pannexin 1 forms ion and metabolite permeable hexameric channels with abundant expression in the central nervous system and elsewhere. Although pannexin 1 does not form intercellular channels, a common channel topology and oligomerization state, as well as involvement of the intracellular carboxyl terminal (CT) domain in channel gating, is shared with connexins. In this study, we characterized the secondary structure of the mouse pannexin 1 cytoplasmic domains to complement structural studies of the transmembrane segments and compare with similar domains from connexins. A combination of structural prediction tools and circular dichroism revealed that, unlike connexins (predominately intrinsically disordered), cytosolic regions of pannexin 1 contain approximately 50% secondary structure, a majority being α-helical. Moreover, prediction of transmembrane domains uncovered a potential membrane interacting region (I360-G370) located upstream of the caspase cleavage site (D375-D378) within the pannexin 1 CT domain. The α-helical content of a peptide containing these domains (G357-S384) increased in the presence of detergent micelles providing evidence of membrane association. We also purified a pannexin 1 CT construct containing the caspase cleavage site (M374-C426), assigned the resonances by NMR, and confirmed cleavage by Caspase-3 in vitro. On the basis of these structural studies of the cytoplasmic domains of pannexin 1, we propose a mechanism for the opening of pannexin 1 channels upon apoptosis, involving structural changes within the CT domain.     

Structural and posttranslational analysis of human calcium-binding protein,spermatid-associated 1

Recently, we detected a novel biomarker in human saliva called calcium-binding protein, spermatid-associated 1 (CABS1). CABS1 protein had previously been described only in testis, and little was known of its characteristics other than it was considered a structurally disordered protein. Levels of human CABS1 (hCABS1) in saliva correlate with stress, whereas smaller sized forms of hCABS1 in saliva are associated with resilience to stress. Interestingly, hCABS1 also has an anti-inflammatory peptide sequence near its carboxyl terminus, similar to that of a rat prohormone, submandibular rat 1. We performed phylogenetic and sequence analysis of hCABS1. We found that from 72 CABS1 sequences currently annotated in the National Center for Biotechnology Information protein database, only 14 contain the anti-inflammatory domain “TxIFELL,” all of which are primates. We performed structural unfoldability analysis using PONDER and FoldIndex and discovered three domains that are highly disordered. Predictions of three-dimensional structure of hCABS1 using RaptorX, IonCom, and I-TASSER software agreed with these findings. Predicted neutrophil elastase cleavage density also correlated with hCABS1 regions of high structural disorder. Ligand binding prediction identified Ca²⁺, Mg²⁺, Zn²⁺, leucine, and thiamine pyrophosphate, a pattern observed in enzymes associated with energy metabolism and mitochondrial localization. These new observations on hCABS1 raise intriguing questions about the interconnection between the autonomic nervous system, stress, and the immune system. However, the precise molecular mechanisms involved in the complex biology of hCABS1 remain unclear. We provide a detailed in silico analysis of relevant aspects of the structure and function of hCABS1 and postulate extracellular and intracellular roles.     

Learning a functional grammar of protein domains using natural language word embedding techniques

In this paper, using Word2vec, a widely-used natural language processing method, we demonstrate that protein domains may have a learnable implicit semantic “meaning” in the context of their functional contributions to the multi-domain proteins in which they are found. Word2vec is a group of models which can be used to produce semantically meaningful embeddings of words or tokens in a fixed-dimension vector space. In this work, we treat multi-domain proteins as “sentences” where domain identifiers are tokens which may be considered as “words.” Using all InterPro (Finn et al. 2017) pfam domain assignments we observe that the embedding could be used to suggest putative GO assignments for Pfam (Finn et al. 2016) domains of unknown function.     

Different coexpressions of arthritis-relevant genes between different body organs and different brain regions in the normal mouse population

Structural changes in different parts of the brain in rheumatoid arthritis (RA) patients have been reported. RA is not regarded as a brain disease. Body organs such as spleen and lung produce RA-relevant genes. We hypothesized that the structural changes in the brain are caused by changes of gene expression in body organs. Changes in different parts of the brain may be affected by altered gene expressions in different body organs. This study explored whether an association between gene expressions of an organ or a body part varies in different brain structures. By examining the association of the 10 most altered genes from a mouse model of spontaneous arthritis in a normal mouse population, we found two groups of gene expression patterns between five brain structures and spleen. The correlation patterns between the prefrontal cortex, nucleus accumbens, and spleen were similar, while the associations between the other three parts of the brain and spleen showed a different pattern. Among overall patterns of the associations between body organs and brain structures, spleen and lung had a similar pattern, and patterns for kidney and liver were similar. Analysis of the five additional known arthritis-relevant genes produced similar results. Analysis of 10 nonrelevant-arthritis genes did not result in a strong association of gene expression or clearly segregated patterns. Our data suggest that abnormal gene expressions in different diseased body organs may influence structural changes in different brain parts.     

Highly covarying residues have a functional role in antibody constant domains

The ability to generate and design antibodies recognizing specific targets has revolutionized the pharmaceutical industry and medical imaging. Engineering antibody therapeutics in some cases requires modifying their constant domains to enable new and altered interactions. Engineering novel specificities into antibody constant domains has proved challenging due to the complexity of inter‐domain interactions. Covarying networks of residues that tend to cluster on the protein surface and near binding sites have been identified in some proteins. However, the underlying role these networks play in the protein resulting in their conservation remains unclear in most cases. Resolving their role is crucial, because residues in these networks are not viable design targets if their role is to maintain the fold of the protein. Conversely, these networks of residues are ideal candidates for manipulating specificity if they are primarily involved in binding, such as the myriad interdomain interactions maintained within antibodies. Here, we identify networks of evolutionarily‐related residues in C‐class antibody domains by evaluating covariation, a measure of propensity with which residue pairs vary dependently during evolution. We computationally test whether mutation of residues in these networks affects stability of the folded antibody domain, determining their viability as design candidates. We find that members of covarying networks cluster at domain‐domain interfaces, and that mutations to these residues are diverse and frequent during evolution, precluding their importance to domain stability. These results indicate that networks of covarying residues exist in antibody domains for functional reasons unrelated to thermodynamic stability, making them ideal targets for antibody design. Proteins 2013. © 2012 Wiley Periodicals, Inc.