Prestin Prestin BioinformaticsBioinformatics

Prestin LocationPrestin Location

Dallos ‘02

Slc26 Family MemberSlc26 Family Member

Mount ‘04

Okoruwa ‘08

Current Functional Hypothesis:Current Functional Hypothesis:

Incomplete TransporterIncomplete Transporter

Dallos ‘02

Primary Structure Clues:Primary Structure Clues:

Slc26 DomainsSlc26 Domains

o STAS (sulfate transporter and anti-sigma factor antagonist)

o ASA (anti-sigma factor atagonists – SpoIIAA 1AUZ)

~σASA-P

ASA

σ

o Unknown role in Slc26

o Mutations result in disease (Slc26a4)

Primary Structure Clues:Primary Structure Clues:

Sulfate Transport Motif Sulfate Transport Motif (108-130)(108-130)

((PROSITE PS01130)

Primary Structure Clues:Primary Structure Clues:

Other MotifsOther Motifs

MotifMotif ResiduResiduee

VerifiedVerified

N-Linked Glycosylation N-Linked Glycosylation (N-X-S/T/C)(N-X-S/T/C)

N163, N163, N166N166

Zheng ’01 +Zheng ’01 +

cAMP/cGMP phosophorolation cAMP/cGMP phosophorolation sitesite

S238, S238, T560T560

Deak ‘05Deak ‘05

Tyrosine Containing (YXXΦ)Tyrosine Containing (YXXΦ) Y520, Y520, Y526Y526

Zheng ‘05Zheng ‘05

Negative ClusterNegative Cluster 557-580557-580 NANA

Positive ClusterPositive Cluster 595-614595-614 NANA

Conservation of DomainsConservation of Domains

Okoruwa ‘08

Secondary/Tertiary Structure Clues:Secondary/Tertiary Structure Clues:

Epitopes and SCAMEpitopes and SCAM

TestTest ResidueResidue ResultResultss

VerifiedVerified

Xpress EpitopeXpress Epitope C TerminalC Terminal ICIC Zheng ’01Zheng ’01

V5 EpitopeV5 Epitope N TerminalN Terminal ICIC Zheng ’01Zheng ’01

AntibodyAntibody 1-201-20 ICIC Zheng ’01Zheng ’01

SCAMSCAM C124C124 ECEC UnpubUnpub

HA TagHA Tag N168N168 ICIC Navaratnam Navaratnam ‘05‘05

SCAMSCAM A170A170 ECEC UnpubUnpub

AntibodyAntibody 274-290274-290 ECEC Navaratnam Navaratnam ‘05‘05

AntibodyAntibody 359-379359-379 ECEC Navaratnam Navaratnam ‘05‘05

SCAMSCAM G366G366 ECEC UnpubUnpub

Secondary/Tertiary Structure Clues:Secondary/Tertiary Structure Clues:

Proposed Topology Proposed Topology

## ResidueResidue StructureStructure ## ResidueResidue StructureStructure

11 C-TermC-Term Xpress Epitope InaccessibleXpress Epitope Inaccessible 99 274-290274-290 Antibody AccessibleAntibody Accessible

22 1-201-20 Antibody InaccessibleAntibody Inaccessible 1010 359-376359-376 Antibody AccessibleAntibody Accessible

33 109-130109-130 Sulfate Transporter MotifSulfate Transporter Motif 1111 G366G366 SCAM AccessibleSCAM Accessible

44 C124C124 SCAM AccessibleSCAM Accessible 1212 Y520, Y520, Y526Y526

Tyrosine Containing MotifTyrosine Containing Motif

55 N163, N163, N166N166

N-Linked GlycosylationN-Linked Glycosylation 1313 557-580557-580 Negative ClusterNegative Cluster

66 A170A170 SCAM AccessibleSCAM Accessible 1414 595-614595-614 Positive ClusterPositive Cluster

77 193-503193-503 Sulfate Transporter DomainSulfate Transporter Domain 1515 635-709635-709 STAS DomainSTAS Domain

88 S238, S238, T560T560

cAMP/cGMP phosophorylation sitecAMP/cGMP phosophorylation site 1616 N-TermN-Term V5 Epitope InaccessibleV5 Epitope Inaccessible

Note: N168 HA Tag Excluded

General Structural General Structural HypothesesHypotheses

Mio ‘07

Point MutationsPoint Mutations Functional AffectFunctional Affect

Type Amino Acids MT OligoNLC Peak

(ΔmV)NLC

(Norm) Author(s) Notes

N-Terminal Deletion 1-20 NM Disrupted 30 0.250 Navaratnam '05  

Point Mutation A100W NM NM FL FL Rajagopalan '06  

Point Mutation A102W NM NM FL FL Rajagopalan '06  

Point Mutation A104W NM NM FL FL Rajagopalan '06  

Multiple Mutations C1 (K233Q, K235Q, R236Q) NM NM -100 NA Oliver '01  

Multiple Mutations C4 (R571Q, R572Q, K577Q) NM NM 25 NA Oliver '01  

C-Terminal Deletion 713-744 NM NM 0 0.125 Navaratnam '05  

Chimera gPres/PAT1551-744 II NM NM NM Zheng '05  

Chimera gPres/PDS644-744 II NM NM NM Zheng '05  

Over 63 mutant, chimeric and insert prestin clones have been created and tested.

Known TechniquesKnown Techniques

Blast (NCBI)Blast (NCBI) Alignment (STRAP/CLC)Alignment (STRAP/CLC) Conserved Domains (NCBI)Conserved Domains (NCBI) Phylogeny (NCBI/CLC)Phylogeny (NCBI/CLC) Hydrophobicity (STRAP)Hydrophobicity (STRAP)

Where Bioinformatics Where Bioinformatics May HelpMay Help

Structural MotifsStructural Motifs DomainsDomains TopologyTopology Secondary/Tertiary StructureSecondary/Tertiary Structure

Transporter Transporter Motor Protein Transition Motor Protein Transition + All conserved residues in mammalian Slc26a5+ All conserved residues in mammalian Slc26a5 - All conserved residues in non-mammalian Slc26a5- All conserved residues in non-mammalian Slc26a5

Oligomerization MotifOligomerization Motif Homoligomerization (tetramer?)Homoligomerization (tetramer?) HeteroligomerizationHeteroligomerization Interactome (pillar protein, phos/dephos)Interactome (pillar protein, phos/dephos)

Modification MotifModification Motif Post-translation ModificationsPost-translation Modifications Phosphorylation SitesPhosphorylation Sites

Plasma Membrane TargetingPlasma Membrane Targeting

Identification of Possible Identification of Possible Oligomerization SiteOligomerization Site