Biology subjects

Leaman, D. P.

Publications and source records attributed to Leaman, D. P..

6 recordsLinked to original sources

bioRxiv2026

The membrane distal domain of CD16a allosterically regulates NK cell ADCC

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is mediated by the activating IgG receptor CD16a (Fc{gamma}RIIIa), yet the molecular mechanisms governing receptor activation remain poorly understood. We demonstrate that the membrane-distal domain 1 (D1) of CD16a functions as an allosteric checkpoint that controls ADCC independently of IgG-Fc binding. A nanobody, C28, that binds an electronegative patch in D1 dose-dependently blocks NK cell ADCC against multiple therapeutic antibodies without affecting direct cytotoxicity. A second nanobody, C21, binding an adjacent D1 epitope has no such effect. Cryo-EM structures of the CD16a-IgG-nanobody complex reveal that C28 allosterically competes with core-fucosylated IgG and stabilizes a closed D1 conformation resembling unliganded receptor, even when Fc is bound. Molecular dynamics simulations show that occupation of the D1 epitope rigidifies the IgG-binding site, stabilizing CD16a overall in contrast with IgG binding alone. The nanobody C28 restricts CD3{zeta} phosphorylation in both resting and ADCC-activated NK cells, revealing tonic inhibitory control upstream of the signaling cascade. Using MINFLUX nanoscopy, we also show that CD16a forms dimers of [~]9 nm spacing on the NK cell surface, a geometry unaltered by the ADCC-enhancing L48H polymorphism. Drawing on structural parallels with the IgE receptor Fc{varepsilon}RI, which is held inactive as a cholesterol-stabilized dimer, we propose that CD16a dimerization through D1 contacts represents a conserved autoinhibitory mechanism among Fc receptors. Consistent with this model, structure-guided disruption of the C28 epitope in NK-92 cells enhances ADCC potency and killing kinetics, providing a blueprint for engineering improved cellular immunotherapeutics.

immunology↗

bioRxiv2026

Reconstructing a Missing Link of HIV-1 Assembly: HIV-1 Envelope-Matrix Interactions in a Native Viral Context

The envelope surface glycoprotein (Env) on HIV-1 drives cell entry and genome delivery through its receptor binding and membrane fusion activities. Incorporation of Env onto assembling virions is governed by its cytoplasmic tail (Env-CT) and the matrix (MA) domain of the PR55Gag (Gag) polyprotein. To better understand how Gag recruits Env onto virions while reducing its antigenic profile by restricting Env copy number to low levels, we used cryo-electron tomography (cryo-ET) and subtomogram averaging combined with molecular dynamics simulations to investigate Env-MA association in intact viral particles. Full-length Env-CT was resolved directly over individual MA trimers in regions of MA lattice discontinuity. We observed that the conserved but enigmatic Kennedy-sequence motif in Env-CT forms a key linkage between Env and MA. Mutational analysis confirmed the impact of specific CT and MA interactions on Env incorporation. Gag maturation released MA lattice restraints on Env, facilitating its clustering and promoting fusion activity.

biochemistry↗

bioRxiv2026

Structure of HIV-1 Env glycoprotein on virions reveals an alternative fusion subunit organization and native membrane coupling

An effective vaccine for Human Immunodeficiency Virus type-1 (HIV-1) has yet to be developed, and detailed characterization of functional Env glycoprotein, the primary antigenic target on virions, has remained elusive. While engineered Env trimers recapitulate many aspects of functional Env, key differences in antigenicity and dynamic behavior have been reported. Here, cryo-electron tomography and subtomogram averaging of HIV-1 virus-like particles (VLPs) revealed conformational differences in critical membrane-proximal regions compared to soluble Envs. Hydrogen/Deuterium-Exchange Mass Spectrometry and Molecular Dynamics captured dynamic profiles of membrane-bound Env and identified critical interactions with membrane. We show that disruption of the viral membrane results in relaxation of Env to a form that resembles engineered, soluble trimers. Additionally, Env from mature and immature VLPs exhibit only minor conformational differences, while surface clustering on virions changes significantly. These studies provide new insights into the essential role the membrane plays in maintaining Env in its native conformational form.

biochemistry↗

bioRxiv2024

Spatial localization of CD16a at the human NK cell ADCC lytic synapse

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc{gamma}RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.

immunology↗

bioRxiv2021

Cryo-ET of HIV reveals Env positioning on Gag lattice and structural variation among Env trimers

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1[A] sub-tomogram averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers plus a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

biophysics↗

bioRxiv2020

Membrane Env liposomes for immunization with HIV spikes

A key goal in HIV vaccine design remains to elicit broadly neutralizing antibodies (bnAbs) against the membrane-embedded envelope glycoprotein spike (mEnv). However, mEnv has lagged behind engineered soluble Envs in vaccine development due to low expression yields and the presence of extraneous proteins on particles. Here, we describe a mEnv vaccine platform that requires no extra proteins or protein engineering. MEnv trimers were fixed, purified and combined with liposomes in mild detergent. On removal of detergent, mEnvs were observed embedded in particles, designated mEnv liposomes (MELs), which were recognized by HIV bnAbs but not non-nAbs. Following sequential immunization in rabbits, MEL antisera neutralized select tier 2 HIV isolates. Variations between the Env immunogens, including a missing N-glycosylation site at position 197 near the CD4 binding site, provide insights into the specificities elicited and possible ways to improve immunogens. MELs can facilitate vaccine design to elicit HIV bnAbs using biochemically defined and multimerized mEnv.

immunology↗