Anastasia Nikolskaya Lai-Su Yeh Protein Information Resource Georgetown University Medical Center

Anastasia Nikolskaya Lai-Su Yeh

Protein Information ResourceGeorgetown University Medical CenterWashington, DC

PIR: a comprehensive resource for functional analysis of protein sequences and families

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PIR Web Site NEW web site, soon to become publichttp://pir.georgetown.edu currently an old version

PIR and UniProt web sites interlinked and cross-navigable

PIR-specific features

Text Search Sequence Search Classification Database Search

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i

• Integration of protein family, function, structure

• Rich links (executive summary + hypertext links) to > 90 databases

• Value-added reports for 1.96 Million UniProtKB protein entries

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iProClass Protein Knowledgebase

Disease/Variation

OMIMHapMap

…Ontology

GO

Protein Sequence

UniProtUniRefUniParcRefSeq

GenPept…

Gene/Genome

GenBank/EMBL/DDBJLocusLinkUniGene

MGITIGR

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Gene Expression

GEOGXD

ArrayExpressCleanExSOURCE

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Structure

PDBSCOPCATH

PDBSumMMDB

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Family

PIRSFInterPro

PfamPrositeCOG

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Interaction

DIPBIND

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Taxonomy

NCBI TaxonNEWT

Protein Expression

Swiss-2DPAGEPMG

…

Literature

PubMed

Function/Pathway

EC-IUBMBKEGG

BioCartaEcoCyc

WIT…

Modification

RESIDPhosphoBase

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iProClass

Integrated Protein Knowledgebase

iProClass

Integrated Protein Knowledgebase

http://pir.georgetown.edu/iproclass

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Example

Want to find info on chorismate mutases,Specifically:Start with Bacillus subtilis P19080 = CHMU_BACSU

Relatedness to other chorismate mutases- Homology- Domain architecture

- Is it related to E.coli P07022 (a well-studied bifunctional enzyme (P-protein), chorismate mutase/prephenate dehydratase)

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iProClass Sequence Report

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What can we find about “chorismate mutase”

Protein Analysis: I. Text Search iProClass

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Text SearchResults (I)

UniProt ID

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Text SearchResults (II)

Display options: add or remove columns

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Text Search Results (III)

Find chorismate mutase(s) from B. subtilis

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Determining Protein HomologyIs B. subtilis CM P19080 homologous to E.coli P-protein P07022? to B. subtilis AroA(G) P39912?Which domains, if any, in multidomain chorismate mutases it corresponds to?What kinds of domain architecture exist in chorismate mutases?

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Retrieve Proteins by UID in Batch Mode

ID mapping option: can use various non-UniProt IDs

Batch Retrieval

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Determining Protein Homology:Sequence Search

BLAST FASTA SSearch

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Blast Search ResultsBLAST query UniProt sequence P19080hits PIRSF005965 family members as best hits

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Pre-compiled Related Sequences: saves time

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BLAST/SSEARCH Results

SSEARCH Alignment

BLASTAlignment

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Determining Protein Homology: Peptide Search

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Peptide Search Results

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Protein families reflect evolutionary relationships Function often follows along the family lines Therefore, matching a protein sequence a protein family

provides information about a protein (need a highly curated and annotated family)

Faster and often more accurate than searching against a protein database

Protein classification facilitates sequence and functional analysis of proteins and is used for accurate automatic annotation (PIRSF is used for UniProt annotation)

Family Classification System:One-Stop Platform for Protein Analysis

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PIRSF Classification System PIRSF: reflects evolutionary relationships of full-length

proteins

Definitions: Basic unit = Homeomorphic Family Homologous: Inferred by sequence similarity Homeomorphic: Full-length sequence similarity and common domain

architecture Hierarchy: Flexible number of levels with varying degrees of sequence

conservation; Network Structure: multiple domain parents

Advantages: Annotation of both generic biochemical and specific biological functions Accurate propagation of annotation and development of standardized

protein nomenclature and ontology

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PIRSF001499: Bifunctional CM/PDH (T-protein)

PIRSF006786: PDH, feedback inhibition-insensitive

PIRSF005547: PDH, feedback inhibition-sensitive

PF02153: Prephenatedehydrogenase (PDH)

PIRSF017318: CM of AroQ class, eukaryotic type

PIRSF001501: CM of AroQ class, prokaryotic type

PIRSF026640: Periplasmic CM

PIRSF001500: Bifunctional CM/PDT (P-protein)


PF01817: Chorismatemutase (CM)

PIRSF006493: Ku, prokaryotic type

PIRSF500001: IGFBP-1

…


PIRSF Homeomorphic Subfamily

• 0 or more levels

• Functional specialization

PIRSF018239: IGFBP-related protein, MAC25 type

PIRSF001969: IGFBP

PIRSF003033: Ku70 autoantigen


PIRSF Homeomorphic Family• Exactly one level

• Full-length sequence similarity and common domain architecture

PIRSF Superfamily


• One or more common domains

PF00219: Insulin-like growth factor binding protein

(IGFBP)

PIRSF800001: Ku70/80 autoantigenPF02735: Ku70/Ku80 beta-barrel domain

Domain Superfamily• One common Pfam

domain


PIRSF006786: PDH, feedback inhibition-insensitive

PIRSF005547: PDH, feedback inhibition-sensitive

PF02153: Prephenatedehydrogenase (PDH)

PIRSF017318: CM of AroQ class, eukaryotic type

PIRSF001501: CM of AroQ class, prokaryotic type

PIRSF026640: Periplasmic CM

PIRSF001500: Bifunctional CM/PDT (P-protein)


PF01817: Chorismatemutase (CM)

PIRSF006493: Ku, prokaryotic type


…


PIRSF Homeomorphic Subfamily


• Functional specialization

PIRSF018239: IGFBP-related protein, MAC25 type

PIRSF001969: IGFBP



PIRSF Homeomorphic Family• Exactly one level

• Full-length sequence similarity and common domain architecture

PIRSF Superfamily


• One or more common domains

PF00219: Insulin-like growth factor binding protein

(IGFBP)

PIRSF800001: Ku70/80 autoantigenPF02735: Ku70/Ku80 beta-barrel domain

Domain Superfamily• One common Pfam

domain

PIRSF Classification SystemA protein may be assigned to only one homeomorphic family, which may have zero or more child nodes and zero or more parent nodes. Each homeomorphic family may have as many domain superfamily parents as its members have domains.

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Unclassified UniProtKB proteins

Uncurated Homeomorphic Clusters

Orphans

Preliminary Homeomorphic Families

Final Families, Subfamilies, Superfamilies

Add/Remove Members

Name, Refs, Abstract, Domain Arch.

Automatic Clustering

Computer-assisted Manual Curation

Automatic Procedure Unassigned Proteins

Au

tom

atic

Pla

ce

me

nt

Hierarchies (Superfamilies/Subfamilies)

Map Domains on Clusters

Merge/Split Clusters

New Proteins

Protein Name Rules/Site Rules Build and Test HMMs

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Orphans



Add/remove members

Name, refs, abstract, domain arch.

Automatic clustering


Automatic Procedure Unassigned proteins

Au

tom

atic

pla

ceme

nt

Hierarchies (superfamilies/subfamilies)

Map domains on Clusters

Merge/splitclusters

New proteins

Protein Name Rule/Site Rule Build and test HMMs

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2

3

4

5

6

7 8



Orphans



Add/remove members

Name, refs, abstract, domain arch.

Automatic clustering


Automatic Procedure Unassigned proteins

Au

tom

atic

pla

ceme

nt

Hierarchies (superfamilies/subfamilies)

Map domains on Clusters

Merge/splitclusters

New proteins

Protein Name Rule/Site Rule Build and test HMMs

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2

3

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7 8

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Tool: Curator’s Decision Maker

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Classification Tool: BlastClust Curator-guided

clustering

Single-linkage clustering using BLAST

Retrieve all proteins sharing a common domain

Iterative BlastClust (fixed length coverage)

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Family Analysis of Homologous Proteins1. Fully Curated Protein Family:

Especially important when the protein of interest is underannotated or misannotated (happens often!)

Evidence types: Characterized (validated), Predicted (by computational methods) or Uncharacterized

2. Preliminary or Uncurated Family Have to do some analysis OR contact PIR and ask to prioritize this family

3. No Family Classification Have to do some analysis OR contact PIR and ask to prioritize this family

iProClass search PIRSF - blank

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Underannotated Proteins

Search iProClass with PIRSF005965

Providing more information

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PIRSF SCAN (sequence search)

UniProt sequence Q8Y5X7 is automatically classified as chorismate mutase of the AroH classPIRSF005965

Returns only matches to fully curated PIRSFs

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Taxonomic distribution of PIRSF can be used to infer evolutionary history of the proteins in the PIRSF

PIRSF Family Report: Curated Protein Family Information

Phylogenetic tree and alignment view allows further sequence analysis

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PIRSF Family Report (II)

Integrated value added information from other databases

Mapping to other protein classification databases

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CM from B.subtilis P19080 does not bring B.subtilis AroA(G) or E. coli P-protein (or related proteins) in BLAST search

Contains a different PFAM domain Identical conserved motifs are not found NOT homologous

PIRSF reports: abstracts contain most of this info PIRSF domain architecture (curated or uncurated): Pfam and

newly defined domains Structure information (PDB links) Hierarchy in DAG (under development)

Chorismate Mutase Results from iProClass Analysis

Use PIRSF family database for the same analysis:

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PIRSF Text Search

New domain

AroA(G)

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Chorismate Mutase Convergent Evolution – EC 5.4.99.5 (Non-Orthologous Gene

Displacement) Two Distinct Sequence/Structure Types

AroQ Class: SCOP (all ), core: 6 helices, bundle AroH Class: SCOP (+), core: beta-alpha-beta-alpha-beta(2)

Two Pfam Domains: PF01817, PF07736 (New PFAM domain)

AroQAroQ AroHAroH

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Developing DAG Viewer

Before:all chorismate mutase proteins and families hit PF01817includingPIRSF005965(not homologous to the rest)

Subfamily

Network structure (in DAG) for PIRSF family classification system reflects PIRSF family hierarchy which is based on evolutionary relationships

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DAG Viewer (II)

After:PFAM created a new domain PF07736which is found in PIRSF005965 members

“Orphans”: no family classification

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PIR Team Dr. Cathy Wu, Director

Protein Classification teamDr. Winona Barker Dr. Lai-Su Yeh Dr. Anastasia NikolskayaDr. Darren Natale Dr. Zhang-Zhi Hu Dr. Raja Mazumder Dr. CR Vinayaka Dr. Sona Vasudevan Dr. Cecilia Arighi

Informatics teamDr. Hongzhan Huang Dr. Peter McGarvey Baris Suzek, M.S. Sehee Chung, M.S. Dr. Leslie Arminski Dr. Hsing-Kuo Hua Yongxing Chen, M.S. Jian Zhang, M.S. Dr. Xin Yuan

Students

Christina Fang Vincent Hermoso Natalia Petrova

UniProt is supported by the National Institutes of Health, grant # 1 U01 HG02712-01

Anastasia Nikolskaya Lai-Su Yeh Protein Information Resource Georgetown University Medical Center

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