Structural Characterization of Semen Coagulum-Derived SEM1(86–107) Amyloid Fibrils That Enhance HIV-1 Infection

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

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Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt Downstream Conformations That Remain Responsive to Conformation-Preferring Ligands

Journal of Virology ◽

10.1128/jvi.00597-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Maolin Lu ◽

Xiaochu Ma ◽

Nick Reichard ◽

Daniel S. Terry ◽

James Arthos ◽

...

Keyword(s):

Structural Characterization ◽

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Conformational State ◽

Broadly Neutralizing Antibodies ◽

Membrane Bound ◽

State 1 ◽

Hiv 1 ◽

Conformation State

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated “SOS” (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env. IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.

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Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers

Retrovirology ◽

10.1186/1742-4690-11-42 ◽

2014 ◽

Vol 11 (1) ◽

pp. 42 ◽

Cited By ~ 8

Author(s):

Philipp Arnold ◽

Patricia Himmels ◽

Svenja Weiß ◽

Tim-Michael Decker ◽

Jürgen Markl ◽

...

Keyword(s):

Structural Characterization ◽

Hiv 1

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Structural Characterization of the HIV-1 Vpr N Terminus

Journal of Biological Chemistry ◽

10.1074/jbc.m305413200 ◽

2003 ◽

Vol 278 (44) ◽

pp. 43188-43201 ◽

Cited By ~ 42

Author(s):

Karsten Bruns ◽

Torgils Fossen ◽

Victor Wray ◽

Peter Henklein ◽

Uwe Tessmer ◽

...

Keyword(s):

Structural Characterization ◽

N Terminus ◽

Hiv 1

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Exploring the structure and formation mechanism of amyloid fibrils by Raman spectroscopy: a review

The Analyst ◽

10.1039/c5an00342c ◽

2015 ◽

Vol 140 (15) ◽

pp. 4967-4980 ◽

Cited By ~ 96

Author(s):

Dmitry Kurouski ◽

Richard P. Van Duyne ◽

Igor K. Lednev

Keyword(s):

Raman Spectroscopy ◽

Formation Mechanism ◽

Structural Characterization ◽

Amyloid Fibrils ◽

Label Free ◽

Amyloidogenic Proteins ◽

Non Destructive

Applications of Raman spectroscopy, a label-free non-destructive technique, for the structural characterization of amyloidogenic proteins, prefibrilar oligomers, and mature fibrils.

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Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1

RNA ◽

10.1261/rna.7205405 ◽

2005 ◽

Vol 11 (6) ◽

pp. 873-884 ◽

Cited By ~ 54

Author(s):

A. K. DEY

Keyword(s):

Structural Characterization ◽

Rna Aptamer ◽

Hiv 1

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Functional and Structural Characterization of HIV-1 gp41 Ectodomain Regions in Phospholipid Membranes Suggests that the Fusion-active Conformation Is Extended

Journal of Molecular Biology ◽

10.1016/j.jmb.2006.08.091 ◽

2006 ◽

Vol 364 (5) ◽

pp. 1103-1117 ◽

Cited By ~ 29

Author(s):

Ofir Korazim ◽

Kelly Sackett ◽

Yechiel Shai

Keyword(s):

Structural Characterization ◽

Phospholipid Membranes ◽

Active Conformation ◽

Gp41 Ectodomain ◽

Hiv 1

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High Resolution Structural Characterization of Aβ42 Amyloid Fibrils by Magic Angle Spinning NMR

Journal of the American Chemical Society ◽

10.1021/jacs.5b03997 ◽

2015 ◽

Vol 137 (23) ◽

pp. 7509-7518 ◽

Cited By ~ 66

Author(s):

Michael T. Colvin ◽

Robert Silvers ◽

Birgitta Frohm ◽

Yongchao Su ◽

Sara Linse ◽

...

Keyword(s):

High Resolution ◽

Structural Characterization ◽

Amyloid Fibrils ◽

Magic Angle Spinning ◽

Magic Angle ◽

Angle Spinning

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Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules

10.26434/chemrxiv.13008212 ◽

2020 ◽

Author(s):

Emma Cawood ◽

Nicolas Guthertz ◽

Jessica Ebo ◽

Theodoros Karamanos ◽

Sheena E. Radford FRS ◽

...

Keyword(s):

Protein Interactions ◽

Structural Characterization ◽

Self Assembly ◽

Molecular Mechanisms ◽

Amyloid Fibrils ◽

Fibril Formation ◽

X Ray Crystallography ◽

Starting Point ◽

Amyloid Assembly

Protein-protein interactions (PPIs) are involved in many of life’s essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific intermediates for detailed study. Such information can also provide a starting point for drug discovery. Here, we demonstrate that covalently tethered small molecule fragments can be used to stabilize specific oligomers during amyloid fibril formation, facilitating the structural characterization of these assembly intermediates. We exemplify the power of covalent tethering using the naturally occurring truncated variant (ΔN6) of the human protein β2-microglobulin (β2m), which assembles into amyloid fibrils associated with dialysis-related amyloidosis. Using this approach, we have trapped tetramers formed by ΔN6 under conditions which would normally lead to fibril formation and found that the degree of tetramer stabilization depends on the site of the covalent tether and the nature of the protein-fragment interaction. The covalent protein-ligand linkage enabled structural characterization of these trapped oligomeric species using X-ray crystallography and NMR, providing insight into why tetramer stabilization inhibits amyloid assembly. Our findings highlight the power of “post-translational chemical modification" as a tool to study biological molecular mechanisms.

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