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Academic Background

David Lambright received his BS from the University of Lowell in 1984 and his PhD in chemistry from Stanford University in 1992. He was a Damon Runyon-Walter Winchell postdoctoral fellow from 1992-1995 in the Department of Molecular Biophysics and Biochemistry at Yale University. He joined the University of Massachusetts Medical School as a faculty member in the Program in Molecular Medicine in 1996. He is a recipient of a Scholar Award from the Leukemia & Lymphoma Society of America.

Structural and molecular mechanisms of cell signaling and membrane trafficking

Photo: David 
G. LambrightResearch in this laboratory is concerned with structural and molecular mechanisms of cell signaling and membrane trafficking. Our approach combines a broad range of experimental methods from diverse disciplines including biochemistry, biophysics, X-ray crystallography, and bioinformatics as well as molecular, cell, and systems biology. Areas of interest include the regulation of membrane trafficking by Rab GTPases, phosphoinositide signaling, and the regulation of cell proliferation. Defects in these fundamental regulatory mechanisms play critical roles in complex disease states such as cancer and diabetes as well as genetically linked disorders.

Rab GTPases comprise a large family of molecular switches that function in membrane trafficking and organelle biogenesis by cycling between active (GTP bound) and inactive (GDP bound) states. Activation is regulated by guanine nucleotide exchange factors (GEFs), which promote exchange of GTP for GDP in response to extracellular or intracellular signals. Inactivation is regulated by GTPase-activating proteins (GAPs), which stimulate the hydrolysis of GTP. In the active state, Rab GTPases interact with a diverse effector proteins to regulate vesicle budding, cargo sorting, and motor-dependent transport as well as the tethering, docking, and fusion of vesicles with target membranes. We seek to understand the structural basis underlying the nucleotide dependent interactions of Rab GTPases with effectors and regulatory factors and determine the mechanisms by which these interactions regulate membrane trafficking. Towards this end, we have developed a novel structural proteomic strategy to profile interactions with the Rab family and determine the underlying structural bases. High throughput microplate assays are used to quantitatively profile interactions with GEFs, effectors, and GAPs. In addition to identifying novel interaction partners, the family-wide analyses facilitate crystallographic studies of Rab-GEF, Rab-effector, and Rab-GAP complexes and are also being used to characterize interactions of Rab GTPases with viral and bacterial virulence factors.

Lipid second messengers known as phosphoinositides regulate a broad spectrum of cellular functions including survival, membrane trafficking, cytoskeletal dynamics, and migration. Targets of phosphoinositides include pleckstrin homology (PH) and FYVE domains in modular signaling and trafficking proteins. Our goal is to understand how phosphoinositide binding domains recognize phosphoinositides and elucidate the mechanisms by which phosphoinositides regulate the assembly and activation of multiprotein signaling and trafficking complexes on intracellular membranes.

A third area of interest concerns an evolutionarily conserved protein, Zpr1, which is required for viability, normal cell cycle progression, and cell growth. Zpr1 is retained in the cytoplasm of quiescent cells through interactions with inactive growth factor receptors and, following stimulation, assembles into cytoplasmic complexes with elongation factor 1-alpha (eEF1A) and nuclear complexes with the survival motor neurons (SMN) protein. An exon deletion in the SMN1 gene that disrupts the interaction with Zpr1 is responsible for the most severe form of spinal muscular atrophy known as Werdnig-Hoffmann syndrome. We have solved the crystal structure of Zpr1 and are working on the structural bases underlying the interactions with growth factor receptors, EF1A, and the SMN complex.


GTPases and phosphoinositides

Regulation of intracellular membrane trafficking

The trafficking of lipids, integral membrane proteins and soluble cargo between membrane delimited organelles is regulated by GTPases of the Rab, Arf, Arl and Sar families as well as mono- and polyphosphorylated derivatives of phosphatidyl inositol (PIPs).


Rabs Image  



The Rab GTPase Cycle

As evolutionarily conserved molecular switches, Rab GTPases cycle between inactive (GDP-bound) and active (GTP-bound) states.  In the active state, Rab GTPases interact with structurally and functionally diverse effectors including cargo sorting complexes on donor membranes, motor proteins involved in vesicular transport and tethering complexes that regulate vesicle fusion with acceptor membranes.  Rab GTPases are activated by guanine nucleotide exchange factors (GEFs) and deactivated by GTPase activating proteins (GAPs), which accelerate the slow intrinsic rates of nucleotide exchange and GTP hydrolysis.  Dual prenylation of C-terminal cysteine motifs allows Rab GTPases to partition with membranes.  Transfer between membranes is facilitated by GDP dissociation inhibitor (GDI) and GDI displacement factors (GDFs).  Targeting of Rab GTPases to specific organelles also depends on GEFs, effectors and GAPs.


Rab Cycle Image  


Membrane targeting mechanisms

Lipid binding domains including those that recognize phosphoinositides utilize ligand-specific and/or non-specific mechanisms for partitioning with lipid bilayers.


LBD Image  

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Rab family-wide interaction analyses

Quantitative profiling of interactions with the Rab GTPase family

Quantitative high throughput microplate assays are used to profile interactions of GEFs, GAPs and effectors with Rab GTPases.  Applications include determination of family-wide specificity profiles, identification of novel interaction partners and mutational analyses of specificity determinants.


HTP Image  

Activation of Rab GTPases by Vps9 domain GEFs

Rab specificity profile for the Rabex-5 catalytic core

Rab5 is an essential regulator of endosomal trafficking and endosome biogenesis.  Rabex-5 is a Rab5 GEF with a Vps9 domain homologous to the yeast Vps9 protein implicated in vacuolar protein sorting.  A profile of the Rab specificity of the catalytic core of Rabex-5 revealed equivalently high exchange activity for Rabs 5 and 21, weak activity for Rab22 and no detectable activity for 29 other Rab GTPases.  Rabs 5, 21 and 22 comprise a small phylogenetic subfamily of endosomal Rab GTPases.


Rabex Profile Image  

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Rabex-5 structure and mutational analyses of recognition determinants

The crystal structure of the Rabex-5 catalytic core revealed a tandem architecture consisting of a Vps9 domain stabilized by a helical bundle.  Conserved exchange determinants map to a common surface of the Vps9 domain, which recognizes invariant aromatic residues in the switch regions of Rab GTPases and selects for the Rab5 subfamily by requiring a small nonacidic residue preceding a critical phenylalanine in the switch I region.

Rabex Structure Image  


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Structural basis for Rab GTPase activation by Vps9 domain GEFs

The crystal structure of the RABEX-5 helical bundle-Vps9 tandem in complex with nucleotide free RAB21 (a key intermediate in the exchange reaction pathway) revealed how the VPS9 domain recognizes Rab5 subfamily GTPases  (Rabs 5, 21 and 22), accelerates GDP release by destabilizing the magnesium binding site and subsequently stabilizes the high energy nucleotide free intermediate via an aspartic acid finger that simultaneously engages the P-loop lysine and switch II backbone.


Rabex Rab21 Image  


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Rab-effector recognition

Structural genomic survey of the Rab family

To understand how structural similarity and diversity in the active conformation of Rab GTPases contributes to effector recognition, we conducted a structural genomic survey of the mammalian Rab GTPase family.  The results revealed non-phylogenetic similarity and variability in the active conformations of Rab GTPases.


SG Survey Image  

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Rab specificity profile of the multivalent effector Rabensoyn-5

Surface plasmon resonance (SPR) was used to profile the interaction of the central and C-terminal Rab binding domains (RBDs) of the multivalent endosomal effector Rabenosyn-5 with the active form of 33 Rab GTPases.  Despite similar tertiary structures, the central and C-terminal RBDs recognize distinct subsets of Rab GTPases (Rabs 4 and 14 vs. Rabs 5, 22 and 24).


Rbsn Profile Image  

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Structural basis for Rab GTPase recognition by Rabenosyn-5

A truncation analysis of the multivalent Rab GTPase and PI3P effector Rabenosyn-5 mapped the central and C-terminal Rab binding domains (RBDs) to homologous regions.  Structures of the RBDs in complex with Rab4 and Rab22 revealed a common binding modality in which a structurally similar helical hairpin core in the RBDs engages a structurally similar active conformation of the switch and interswitch regions in the respective Rab GTPases.  Mutational analyses further revealed that the differential specificity of the RBDs is due in part to a non-conserved N-terminal extension (NTE) in RDB1 and in part to compositional differences within the conserved helical hairpin cores.


r4r22rbsn Image  


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Conceptual model for Rab-effector recognition

The model shown here is based on known structures of Rab GTPases alone and in complex with effectors and takes into account the results of mutational analyses.  Structural variability influences the spatial disposition and exposure of the conserved residues, sub-dividing the Rab family into non-phylogenetic subsets that satisfy the structural requirements for effector recognition.  Compositional diversity within the switch/interswitch regions (and in certain cases CDRs) further refines the specificity through enhanced affinity for Rab GTPases with compatible compositions (positive selection) and/or reduced affinity for Rab GTPases with incompatible compositions (negative selection).  The family-wide nature of the recognition process is underscored by the conservation of positive determinants in interacting subsets and negative determinants in non-interacting Rab GTPases.  This model is also applicable to interactions with regulatory/accessory factors.


RR Model Image

Structural basis for recruitment of FIP3 to recycling endosomes

Rab11 regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis.  A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11.  Normal completion of cytokinesis requires a complex between Rab11 and FIP3.  Shown here is the structure of a heterotetrameric complex between constitutively active (GTP-bound) Rab11 and a FIP3 construct that includes the RBD.  Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region.  The RBD overlaps with the coiled coil and extends through the C-terminal hook.  Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement that facilitates the interaction with FIP3.


r11fip3 Image


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De-activation of Rab GTPases by TBC domain GAPs

Rab specificity profile of the Gyp1p TBC domain GAP

TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotes and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis.  A profile of the specificity of the Gyp1p TBC domain revealed high GAP activity for Rab33 in addition to Rab1 (the mammalian homologue of the in vivo Gyp1p substrate Ypt1p).  The identification of Rab33 as a Gyp1p substrate facilitated determination of the structure of a TBC domain-Rab GTPase complex.


gyp2 Profile Image  


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TBC domains accelerate GTP hydrolysis by a dual finger mechanism     

In the crystal structure of the Gyp1p TBC-domain-Rab33-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase.  The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain.  Strong conservation of both catalytic fingers suggests that most TBC-domain GAPs will accelerate GTP hydrolysis by a similar dual-finger mechanism.  These conclusions are supported by mutational and complementation analyses.


gyp1r33 Image  


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Phosphoinositide recognition and membrane targeting

Structural basis for 3-phosphoinositide recognition by PH domains

The pleckstrin homology (PH) domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds PIP3 with high affinity.  The structure of the Grp1 PH domain bound to the head group of PIP3 (IP4) revealed a novel mode of phosphoinositide recognition involving a 20-residue beta hairpin insertion within the beta6/beta7 loop.   The observed mode of recognition involving residues from a conserved basic motif as well as the variable loops surrounding the phosphoinositide binding site explains the high affinity and specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains that lack the hairpin insertion.


grp1 Image  


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Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains

The PH domains of the homologous proteins Grp1, ARNO and Cytohesin-1 bind PIP3 with unusually high selectivity.  Remarkably, splice variants that differ only by the insertion of a single glycine residue in the beta1/beta2 loop exhibit dual specificity for PIP2 and PIP3.  Crystal structures for the dual specificity 'triglycine' variant of the ARNO PH domain in complex with the head groups of PIP2 (IP3) and PIP3 (IP4) revealed the structural basis underlying this dramatic selectivity switch.  Loss of contacts with the beta1/beta2 loop with no significant change in head group orientation accounts for the significant decrease in PIP3 affinity observed for the dual specificity variants.  Conversely, a small increase rather than decrease in affinity for PIP2 is explained by a novel 'rotated' binding mode, in which the glycine insertion alleviates unfavorable interactions with the beta1/beta2 loop. These and other observations supported by systematic mutational analyses suggested a general model for phosphoinositide recognition by PH domains that conserve a 'signature' basic motif.


arno Image  


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Structural basis for PI3P recognition and multivalent endosome targeting by FYVE domains

The localization of the early endosomal marker and tethering factor EEA1 is mediated by a C-terminal region that includes a calmodulin binding motif, a Rab5 binding site and a FYVE domain that selectively binds PI3P.  The crystal structure of the C-terminal region bound to the head group of PI3P (IP2) revealed an organized quaternary assembly consisting of a parallel coiled coil and a dyad-symmetric FYVE domain homodimer. Structural and biochemical observations support a multivalent mechanism for endosomal targeting in which domain organization, dimerization and quaternary structure amplify the weak affinity and modest specificity of head group interactions with conserved residues.


eea1 Image 


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