Noreen R. Gonzales

CDD: a Conserved Domain Database for the functional annotation of proteins

NCBI’s Conserved Domain Database (CDD) is a resource for the annotation of protein sequences with the location of conserved domain footprints, and functional sites inferred from these footprints. CDD includes manually curated domain models that make use of protein 3D structure to refine domain models and provide insights into sequence/structure/function relationships. Manually curated models are organized hierarchically if they describe domain families that are clearly related by common descent. As CDD also imports domain family models from a variety of external sources, it is a partially redundant collection. To simplify protein annotation, redundant models and models describing homologous families are clustered into superfamilies. By default, domain footprints are annotated with the corresponding superfamily designation, on top of which specific annotation may indicate high-confidence assignment of family membership. Pre-computed domain annotation is available for proteins in the Entrez/Protein dataset, and a novel interface, Batch CD-Search, allows the computation and download of annotation for large sets of protein queries. CDD can be accessed via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

CDD: NCBI's conserved domain database

NCBIs CDD, the Conserved Domain Database, enters its 15th year as a public resource for the annotation of proteins with the location of conserved domain footprints. Going forward, we strive to improve the coverage and consistency of domain annotation provided by CDD. We maintain a live search system as well as an archive of pre-computed domain annotation for sequences tracked in NCBIs Entrez protein database, which can be retrieved for single sequences or in bulk. We also maintain import procedures so that CDD contains domain models and domain definitions provided by several collections available in the public domain, as well as those produced by an in-house curation effort. The curation effort aims at increasing coverage and providing finer-grained classifications of common protein domains, for which a wealth of functional and structural data has become available. CDD curation generates alignment models of representative sequence fragments, which are in agreement with domain boundaries as observed in protein 3D structure, and which model the structurally conserved cores of domain families as well as annotate conserved features. CDD can be accessed at http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

CDD: specific functional annotation with the Conserved Domain Database.

NCBIs Conserved Domain Database (CDD) is a collection of multiple sequence alignments and derived database search models, which represent protein domains conserved in molecular evolution. The collection can be accessed at http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, and is also part of NCBIs Entrez query and retrieval system, cross-linked to numerous other resources. CDD provides annotation of domain footprints and conserved functional sites on protein sequences. Precalculated domain annotation can be retrieved for protein sequences tracked in NCBIs Entrez system, and CDDs collection of models can be queried with novel protein sequences via the CD-Search service at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. Starting with the latest version of CDD, v2.14, information from redundant and homologous domain models is summarized at a superfamily level, and domain annotation on proteins is flagged as either ‘specific’ (identifying molecular function with high confidence) or as ‘non-specific’ (identifying superfamily membership only).

CDD: a conserved domain database for interactive domain family analysis

The conserved domain database (CDD) is part of NCBIs Entrez database system and serves as a primary resource for the annotation of conserved domain footprints on protein sequences in Entrez. Entrezs global query interface can be accessed at and will search CDD and many other databases. Domain annotation for proteins in Entrez has been pre-computed and is readily available in the form of ‘Conserved Domain’ links. Novel protein sequences can be scanned against CDD using the CD-Search service; this service searches databases of CDD-derived profile models with protein sequence queries using BLAST heuristics, at . Protein query sequences submitted to NCBIs protein BLAST search service are scanned for conserved domain signatures by default. The CDD collection contains models imported from Pfam, SMART and COG, as well as domain models curated at NCBI. NCBI curated models are organized into hierarchies of domains related by common descent. Here we report on the status of the curation effort and present a novel helper application, CDTree, which enables users of the CDD resource to examine curated hierarchies. More importantly, CDD and CDTree used in concert, serve as a powerful tool in protein classification, as they allow users to analyze protein sequences in the context of domain family hierarchies.

Grafting of “Abbreviated” Complementarity-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the “abbreviated” CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients’ sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.

CDD: conserved domains and protein three-dimensional structure

CDD, the Conserved Domain Database, is part of NCBI’s Entrez query and retrieval system and is also accessible via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. CDD provides annotation of protein sequences with the location of conserved domain footprints and functional sites inferred from these footprints. Pre-computed annotation is available via Entrez, and interactive search services accept single protein or nucleotide queries, as well as batch submissions of protein query sequences, utilizing RPS-BLAST to rapidly identify putative matches. CDD incorporates several protein domain and full-length protein model collections, and maintains an active curation effort that aims at providing fine grained classifications for major and well-characterized protein domain families, as supported by available protein three-dimensional (3D) structure and the published literature. To this date, the majority of protein 3D structures are represented by models tracked by CDD, and CDD curators are characterizing novel families that emerge from protein structure determination efforts.

Size-Distribution Analysis of Proteins by Analytical Ultracentrifugation: Strategies and Application to Model Systems

Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.

CDD/SPARCLE: functional classification of proteins via subfamily domain architectures

NCBIs Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBIs Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

Combined Affinity and Rate Constant Distributions of Ligand Populations from Experimental Surface Binding Kinetics and Equilibria

The present article considers the influence of heterogeneity in a mobile analyte or in an immobilized ligand population on the surface binding kinetics and equilibrium isotherms. We describe strategies for solving the inverse problem of calculating two-dimensional distributions of rate and affinity constants from experimental data on surface binding kinetics, such as obtained from optical biosensors. Although the characterization of a heterogeneous population of analytes binding to uniform surface sites may be possible under suitable experimental conditions, computational difficulties currently limit this approach. In contrast, the case of uniform analytes binding to heterogeneous populations of surface sites is computationally feasible, and can be combined with Tikhonov-Phillips and maximum entropy regularization techniques that provide the simplest distribution that is consistent with the data. The properties of this ligand distribution analysis are explored with several experimental and simulated data sets. The resulting two-dimensional rate and affinity constant distributions can describe well experimental kinetic traces measured with optical biosensors. The use of kinetic surface binding data can give significantly higher resolution than affinity distributions from the binding isotherms alone. The shape and the level of detail of the calculated distributions depend on the experimental conditions, such as contact times and the concentration range of the analyte. Despite the flexibility introduced by considering surface site distributions, the impostor application of this model to surface binding data from transport limited binding processes or from analyte distributions can be identified by large residuals, if a sufficient range of analyte concentrations and contact times are used. The distribution analysis can provide a rational interpretation of complex experimental surface binding kinetics, and provides an analytical tool for probing the homogeneity of the populations of immobilized protein.

Minimizing the Immunogenicity of Antibodies for Clinical Application

The clinical utility of murine monoclonal antibodies has been greatly limited by the human anti-murine antibody responses they effect in patients. To make them less immunogenic, murine antibodies have been genetically engineered to progressively replace their murine content with that of their human counterparts. This review describes the genetic approaches that have been used to humanize murine antibodies, including the generation of mouse-human chimeric antibodies, veneering of the mouse variable regions, and the grafting of murine complementarity-determining regions (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules, while retaining only those murine framework residues deemed essential for the integrity of the antigen-binding site. To minimize the anti-idiotypic responses that could still be evoked by the murine CDRs in humanized antibodies, two approaches have also been described. These are based on grafting onto the human frameworks the ‘abbreviated’ CDRs or only the specificity-determining residues (SDRs), the CDR residues that are involved in antigen interaction. The SDRs are identified through the help of the database of three-dimensional structures of antibody:antigen complexes or by mutational analysis of the antibody-combining site. In addition, we also describe the use of in vitro affinity maturation to enhance the binding affinity of humanized antibodies, as well as the manipulation of framework residues to maximize their human content and minimize their immunogenic potential.