Lisa Henry

The WAVE Regulatory Complex Links Diverse Receptors to the Actin Cytoskeleton

The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here, we have identified a large family of potential WRC ligands, consisting of ∼120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans.

Crystal Structure of the Second LNS/LG Domain from Neurexin 1α: Ca2+ BINDING AND THE EFFECTS OF ALTERNATIVE SPLICING*

Neurexins mediate protein interactions at the synapse, playing an essential role in synaptic function. Extracellular domains of neurexins, and their fragments, bind a distinct profile of different proteins regulated by alternative splicing and Ca2+. The crystal structure of n1α_LNS#2 (the second LNS/LG domain of bovine neurexin 1α) reveals large structural differences compared with n1α_LNS#6 (or n1β_LNS), the only other LNS/LG domain for which a structure has been determined. The differences overlap the so-called hyper-variable surface, the putative protein interaction surface that is reshaped as a result of alternative splicing. A Ca2+-binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca2+-binding site in n1α_LNS#2 has low affinity (Kd ∼ 400 μm). Ca2+ binding ceases to be measurable when an 8- or 15-residue splice insert is present at the splice site SS#2 indicating that alternative splicing can affect Ca2+-binding sites of neurexin LNS/LG domains. Our studies initiate a framework for the putative protein interaction sites of neurexin LNS/LG domains. This framework is essential to understand how incorporation of alternative splice inserts expands the information from a limited set of neurexin genes to produce a large array of synaptic adhesion molecules with potentially very different synaptic function.

Crystal structure of Spot 14, a modulator of fatty acid synthesis

Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14’s action is unknown. Here we present the crystal structure of S14 to 2.65 Å and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.

Mechanism of LDL binding and release probed by structure-based mutagenesis of the LDL receptor.

The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. Several different molecular mechanisms have been proposed for the binding of LDL to LDL-R at neutral plasma pH and for its release at acidic endosomal pH. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. We have used a structure-based site-directed mutagenesis approach to examine 12 residues in the extracellular domain of LDL-R for their effect on LDL binding and release. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. Our results are consistent with an alternative model of LDL-R whereby multiple modules of the extracellular domain interact with LDL at neutral pH, concurrently positioning key residues so that at acidic pH the LDL-R:LDL interactions become unfavorable, triggering release. After LDL release, the closed form of LDL-R may target its return to the cell surface.

The Bacterial Effector VopL Organizes Actin into Filament-like Structures

VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

'MAD'ly phasing the extracellular domain of the LDL receptor: a medium-sized protein, large tungsten clusters and multiple non-isomorphous crystals

The crystal structure of the extracellular domain of the LDL receptor (LDL-R) was determined in a MAD experiment using 12-tungstophosphate clusters as anomalous scatterers. While useful for phasing, the tungsten clusters rendered the crystals radiation-sensitive and non-isomorphous and profoundly altered the diffraction data, causing complications. The work is presented as a case study for phasing a medium-sized protein (700 residues) at low resolution (4 A) with multiple non-isomorphous crystals containing 31 W atoms in the asymmetric unit.

Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

Biochemical Reconstitution of the WAVE Regulatory Complex

The WAVE regulatory complex (WRC) is a 400-kDa heteropentameric protein assembly that plays a central role in controlling actin cytoskeletal dynamics in many cellular processes. The WRC acts by integrating diverse cellular cues and stimulating the actin nucleating activity of the Arp2/3 complex at membranes. Biochemical and biophysical studies of the underlying mechanisms of these processes require large amounts of purified WRC. Recent success in recombinant expression, reconstitution, purification, and crystallization of the WRC has greatly advanced our understanding of the inhibition, activation, and membrane recruitment mechanisms of this complex. But many important questions remain to be answered. Here, we summarize and update the methods developed in our laboratory, which allow reliable and flexible production of tens of milligrams of recombinant WRC of crystallographic quality, sufficient for many biochemical and structural studies.