The B-1 business visitor visa is available for a wide variety of business travellers, including domestic workers who are employed by U.S. citizens or nonimmigrants, trainees (B-1 in lieu of H-3), and high-skilled workers employed by foreign companies (B-1 in lieu of H-1B).
The B-1 visa allows foreign nationals to visit the United States on business but generally prohibits work at a U.S. job.1 It is often used by travelers for short-term trips to the U.S. for meetings, conferences or business transactions. There is no annual cap or numerical limit on the number of B-1 visas available each year. The B-1 visitor is not a work visa, and, generally speaking, foreign workers may not use it to come to the U.S. to perform skilled or unskilled labor.2 However, the B-1 visa is ill defined and frequently used by foreign workers who come to the U.S. to represent a foreign business.3 Indeed, there are several subclasses of business visitors who may perform work in the United States.4 Among the type of individuals who may work on a B-1 visa are domestic workers employed by either U.S. citizens who reside abroad or other nonimmigrants, trainees, and some high-skilled temporary workers who are paid by their foreign employer.5 B-1 domestic workers are required to have an employment contract and a required wage as a prerequisite to getting the visa.6 B-1 trainees and workers with the B-1 in lieu of H-1B distinction, however, are not. None of the B-1 subclasses require the employer to submit a labor certification application or otherwise obtain approval from either the U.S. Department of Labor or the U.S. Department of Homeland Security.7 Even courts have noted that business visitor visas are obtained through a faster and less scrutinized process than the H visas for temporary work or training.8 Moreover, there are no regulations or procedures in place to address or prevent the displacement of U.S. workers.
The number of individuals who are working with B-1 visas is unknown. The government maintains information on the number and nationality of individuals receiving B-1 visas and the number of B-1 visa holders who actually enter the United States. But those numbers are meaningless because, presumably, the vast majority of B-1 visa holders do not work. Nevertheless, the number of B-1 visas issued is not broken down by subclasses that allow work, or any other specific measure, such as industry or occupation. Indeed, publicly available information does not reveal how many B-1 visa holders work, where they work, for what purpose, and for how long. Government officials have in the past indicated that up to 20,000 of the B-1 visas issued annually enable visa holders to engage in paid work.9 The lack of concrete data on these subclasses allowing work is significant considering that each year there are over 35,000 B-1 visas issued and over 3 million B-1 admissions.
Consular officials set the duration of a B-1 domestic worker visa based on the employer’s stay in the United States.31 The initial time may exceed one year as long as the stay “is not indefinite in nature.”32 Indeed, the State Department contemplates a longer stay for certain B-1 domestic workers who are employed by U.S. citizens temporarily stationed in the United States for up to 4 years.33 B-1 visas generally are valid for up to one year with the possibility of extension in six-month increments.34 There is no specified limit to the number of extensions a B-1 domestic worker may receive.
B-1 visitor visas for business are also available for individuals who need training in the United States and who would otherwise qualify for the H-3 visa.35 The trainee must have nonimmigrant intent and be clearly employed and paid by a foreign company. The training program must not be available in the trainee’s home country and the training program generally must be less than six months in duration. Productive work in the United States is allowed if it is incidental to the training itself and if the trainee remains an employee of an overseas company and is paid from a source abroad.36 Furthermore, the trainee should not be placed in a position normally occupied by U.S. workers. Unlike B-1 domestic workers, no employment contract is required for the B-1 in lieu of H-3 subclass and no prevailing wage is required, i.e. they are paid the regular wages in their home country while being trained in the United States.37 The B-1 visa is not intended to be a way to avoid the more time-consuming H-3 visa process. There is no limit to the number of B-1 visas. Neither the U.S. State Department nor the U.S. Department of Homeland Security publishes the number of B-1 in lieu of H-3 visas issued or the nationality of these workers.
Immigration regulations state that B-1 visas are initially valid for up to one year with the possibility of six-month extensions.38 With regard to the B-1 in lieu of H-3 visa in particular, however, the U.S. State Department, however, advises that the duration of the B-1 in lieu of H-3 visa should generally be for less than six months.39 However, some practitioners comment that intended stays of more than two weeks will face additional scrutiny during the visa application process.40 Even so, there is no regulation or guidance prohibiting extensions. And there is no apparent limit to the number of possible extensions or maximum duration for visas in this subclass.
B-1 visas are also available for workers in specialized jobs who would otherwise qualify for the H-1B visa and whom a foreign employer pays. They are not intended to displace U.S. workers or to be a way to avoid the more regulated H-1B visa. They are intended to allow foreign nationals who are employed by foreign companies to work temporarily in the United States. Such work often includes performing tasks for U.S. employers who have business relationships with the foreign employer. Unlike B-1 domestic workers, no employment contract is required for the B-1 in lieu of H-1B subclass. Some business immigration lawyers describe it as a flexible choice for international employers who want to avoid the complex and costly H-1B visa process.62 For example, unlike the H-1B program, there is no limit to B-1 visas and employers are not required to meet any prevailing wage requirements. Neither the U.S. State Department nor the U.S. Department of Homeland Security publishes the number of B-1 in lieu of H-1B visas issued or the nationality of these workers.
B-1 visas generally are only valid for up to one year with the possibility of six-month extensions.63 The duration of the initial B-1 in lieu of H-1B visa should be less than six months.64 There is no regulation or guidance prohibiting extensions. Apparently, there is no limit to the number of possible extensions or maximum duration for visas in this subclass.
1. 8 U.S.C. § 1101(a)(15)(B) (a B-1 applicant may not enter the U.S. “for the purpose of . . . performing skilled or unskilled labor”); 22 C.F.R. § 41.31 (a B-1 visa holder may not engage in “local employment or labor for hire”); 9 FAM 41.31 N7(a) (B-1 is not appropriate for “obtaining and engaging in employment” while in the U.S.).
3. M. Jaidi, The Many Legitimate Uses of the B-1 Visa, (April 24, 2011) and C. Mehta and O. Thuma, Analyzing the “B-1” Business Visa, (July 5, 2004), both available at http://www.cyrusmehta.com/News.aspx?SubIdx=ocyrus201142475017&Month=&Fro... (last visited August 2013).
5. There are numerous other subclasses of B-1 visitors for business which may involve work in the United States. See generally 9 FAM 41.31 Notes. Some examples of these subclasses include professional athletes (9 FAM 41.31 N9.4), foreign airline employees (9 FAM 41.31 10.2), and artists (9 FAM 41.31 11.10).
6. 9 FAM 41.31 N9.3.
7. Compare the H visa programs which require approval from both.
8. Chellen v. John Pickle Co., Inc., 344 F. Supp. 2d 1278, 1282 (N.D. Okla. 2004).
9. A. Sukthankar, Global Workers Justice Alliance, Visas, Inc.: Corporate Control and Policy Incoherence in the U.S. Foreign Labor System, p. 11, fn 5 (2012).
22. U.S. State Department, Visitor Visas for Personal or Domestic Employees, available at http://travel.state.gov/visa/temp/types/types_1262.html (August 2013).
23. 9 FAM 41.31 N9.3-2.
24. 9 FAM 41.31 N9.3-3. Nonimmigrants with a B, E, F, H, I, J, L, M, O, P or Q visa are eligible to employ B-1 domestic workers. Legal permanent residents (individuals with a green card), may not employ B-1 domestic workers because by definition they are residing in the U.S. permanently. The B-1 domestic worker visa is meant to be temporary. Cite.
25. 9 FAM 41.31 N9.3-1 a.(5).
26. 9 FAM 41.31 N9.3-1 b; N9.3-2 b.; N9.3-3 a.(4).
27. 9 FAM 41.31 N9.3-5 (source of payment to a B-1 personal or domestic employee or the place where the payment is made or the location of the bank is not relevant).
28. 9 FAM 41.31 N9.3-2 and 9.3-3.
29. 9 FAM 41.31 N9.3.
30. This information may be available through a Freedom of Information Act request directed to the U.S. Citizenship and Immigration Services (USCIS). While the application for an Employment Authorization Document, requires the nonimmigrant to list their visa staus, it may or may not be broken down into the particular B-1 subclass. USCIS does require B-1 domestic workers who apply for extensions on Form I-539 to break down their subclass.
31. 9 FAM 41.112 N2.8, 41.31 N14.4.
32. 9 FAM 41.31 N3.1.
33. 9 FAM 41.31 N9.3-2 b.
34. C. Miller, Ed., U.S. Department of Homeland Security, U.S. Customs and Border Protection Inspector’s Field Manual, at 47, (2008) available at http://www.ilw.com/immigrationdaily/news/2008,0513-cbp.pdf.
35. 9 FAM 41.31 N11.9; 9 FAM 41.53 N4.5; 8 C.F.R. 214.2(h)(7); U.S. State Department, B-1 in Lieu of H, Unclassified Cable 12 State 101466 (Oct. 12, 2012).
36. U.S. State Department, B-1 in Lieu of H, Unclassified Cable 12 State 101466 (Oct. 12, 2012).
37. However, B-1 trainees may be entitled to the minimum wage under the Fair Labor Standards act if in reality they are employees engaging in productive work in the United States. See Chellen v. Pickle, 344 F.Supp. 2d 1278 (N.D. Okla 2004).
38. 8 C.F.R. § 214.2(b)(1); C. Miller, Ed., U.S. Department of Homeland Security, U.S. Customs and Border Protection Inspector’s Field Manual, 2008 at 47, available at http://www.ilw.com/immigrationdaily/news/2008,0513-cbp.pdf.
39. U.S. State Department, B-1 in Lieu of H, Unclassified Cable 12 State 101466 (Oct. 12, 2012).
62. See, e.g., C. Mehta and M. Jaidi, B-1 in Lieu of H-1B Visa in Jeopardy: Don’t Throw the Baby Out with the Bathwater, (May 26, 2011) available at http://cyrusmehta.blogspot.com/2011/05/b-1-in-lieu-of-h-1b-visa-in-jeopa... see also Murphy Law Firm, News Brief: B-1 in Lieu of H-1B – Will this Useful Category Survive?, (July 1, 2011) available at http://www.murthy.com/news/n_b1luh1.html.
63. 8 C.F.R. § 214.2(b)(1); C. Miller, Ed., U.S. Department of Homeland Security, U.S. Customs and Border Protection Inspector’s Field Manual, 2008 at 47, available at http://www.ilw.com/immigrationdaily/news/2008,0513-cbp.pdf.
64. U.S. State Department, B-1 in Lieu of H, Unclassified Cable 12 State 101466 (Oct. 12, 2012). However, according to one immigration law firm, the U.S. embassy in London has made changes in the way it issues B-1 in lieu of H-1 visas. Instead of issuing a visa with an annotation specifying “in lieu of H-1” the practice is now to issue a standard ten-year B-1/B-2 visa (where the applicant qualifies) and enter information regarding the applicant’s B-1 in lieu of H-1 status on the applicant’s records, which are available to CBP officers at Ports of Entry. (The Embassy recommends that B-1 in lieu of H-1 applicants take their full set of documents demonstrating their eligibility on each trip to the U.S.), available at http://www.goldsteinvisa.com/r-changes.html (last visited August 2013).
Employers who hire B-1 nonimmigrants do not petition the government for special permission. Employers are not required to make any application with either the U.S. Department of Labor or the U.S. Department of Homeland Security. Rather, the workers are hired abroad and apply for the visa through the U.S. embassy or consular post.78 Along with the visa application, the worker must present proof of the eligibility for the visa, and be interviewed personally. Because the U.S. State Department does not report statistics for each particular B-1 subclass, the approval rates for domestic workers, trainees (in lieu of H-3) and skilled workers (in lieu of H-1B) are unknown. Once the worker has the visa, he travels to the U.S. and presents for admission at the U.S. border or port of entry. The U.S. Department of Homeland Security’s Customs and Border Protection makes the final decision about whether the individual may enter the United States and for how long he may stay. B-1 domestic workers must apply for an Employment Authorization Document (EAD) from the U.S. Department of Homeland Security, U.S. Citizenship and Immigration Services, before they can start work. B-1 trainees and B-1 in lieu of H-1B workers do not require an EAD, presumably because they are not earning money from within the United States.
Employers locate and hire B-1 workers abroad. There is little information about this process or the extent to which recruiting agencies are involved. In the case of domestic workers, the employer must sign an employment contract before the worker applies for the visa. Some employers are required to pay for the domestic worker’s transportation to and from the United States. Employers of B-1 trainees and B-1 in lieu of H-1B workers are not required to have an employment contract or pay for the worker’s travel to the U.S. However, to obtain the visa, the worker must show that they are employed by the foreign employer and will continue to be paid by their foreign employer and not from any source in the U.S. Presumably, the international employer will be assisting, if not handling outright, the B-1 visa application process for their foreign employees.
The prospective B-1 worker applies for the visa through the U.S. Consulate or embassy abroad. The first step is to complete the Form DS-160 through an online application and upload a photo. The fee to apply for a B-1 visa is $160.79 Generally, B-1 applicants must have an interview at the U.S. embassy or consular post abroad. At the interview, the applicant brings a printed out confirmation of the Form DS-160, a receipt that the fee has been paid, their passport, and any additional documents that show eligibility for the visa, including the purpose of the trip and evidence of a temporary stay in the United States.
Applicants must show they are eligible for the visa and bring all supporting documents to the interview. Domestic workers must present their signed employment contract.80 Individuals applying for B-1 in lieu of H-1B visas should show ongoing work for the overseas employer and clearly show employment with the company or firm abroad.81 If the B-1 applicant is a new employee with the company and their first work assignment is in the U.S., however, it may be difficult to show eligibility for the visa. The worker must prove that payment will not come from a U.S. employer.82 The job or training pursued in the U.S. must be of type, which would qualify the worker for an H-1B visa. In other words, it must be a specialty occupation and the worker must have a bachelor’s degree or equivalent experience. The U.S. Department of Labor has no role in reviewing the job or the applicant.
B-1 domestic workers must obtain an employment authorization document (EAD) prior to starting work for their employer. Workers must submit Form I-765 to the U.S. Citizenship and Immigration Services (USCIS) along with the filing fee of $380.91 Workers may not apply for the EAD until they are present in the United States and it may take up to several months for USCIS to approve the application. Once the worker receives the EAD, she may apply for a Social Security Number and drivers license.
78. Short-term business visitors from an authorized group of about 35 countries also can use the Visa Waiver Program without need to obtain a B-1 visa. Different rules apply for citizens of Mexico and Canada.
79. U.S. State Department, Fees for Visa Services, available at http://travel.state.gov/visa/temp/types/types_1263.html (last visited August 2013).
80. 9 FAM 41.31 N9.3.
81. 9 FAM 41.31 N11. A foreign branch or office of a company based in the U.S. may qualify.
82. 9 FAM 41.31 N11.1; U.S. State Department, Consulate General of the U.S., Mumbai, India, Nonimmigrant Visas: B-1 in Lieu of H-1B, available at http://mumbai.usconsulate.gov/b1_in_lieu_of_h1b.html (last visited August 2013). A U.S. company, however, may provide an expense allowance to the B-1 worker.
83. 9 FAM 41.31 N2.
84. 9 FAM 41.31 N3.4; N4.2.
85. 9 FAM 41.31 N9.3-6 a; see also 8 U.S.C. § 1375c(b)(3), William Wilberforce Trafficking Victims Protection Reauthorization Act of 2008 (WWTVPRA) (including “information on the illegality of slavery, peonage, trafficking in persons, sexual assault, extortion, blackmail, and worker exploitation in the United States.”).
86. Id. Consular officers must add a mandatory case note in the visa applicant computer system stating the pamphlet was provided and the applicant indicated that s/he understood its contents. See also 9 FAM 41.21 N6.5 for information about additional consular officer responsibilities with respect to anti-trafficking measures.
87. 8 U.S.C. §1225; 8 C.F.R. Part 235, Inspection of Persons Applying for Admission; see also Austin T. Fragomen, Jr., Alfred J. Del Rey, Jr., and Sam Bernsen, Immigration Law and Business § 2:11 (2010) (“The issuance of a nonimmigrant visa gives the alien permission to apply for admission to the United States at a port of entry…The visa does not assure an alien that he or she will be admitted to the United States, however; it merely indicates that a consular officer has found the alien eligible for temporary admission to the United States and not inadmissible under § 212(a) of the INA, 8 U.S.C.A. § 1182(a).”).
88. U.S. State Department, B-1 in Lieu of H, Unclassified Cable 12 State 101466 (Oct. 12, 2012) (“These paragraphs were edited in order to clarify activities that will permt a B-1 in lieu of H annotation”).
89. Kehrela Hodkinson, Reggie Pacis and Edward Rios, CBP/Consular Processing Issues, American Immigration Lawyers Association (2012) (noting that “many consular officers are still refusing to annotate” creating “potential issues when the individual arrives at the Port of Entry with an unannotated B-1 and explains that they are coming to work in the U.S.”), available at http://www.ailadownloads.org/seminars/seminar120911resources.pdf (last visited August 2013).
91. U.S. Department of Homeland Security, USCIS, I-765, Application for Employment Authorization, available at http://www.uscis.gov/i-765 (last visited August 2013).
92. See Form I-539 instructions (http://www.uscis.gov/sites/default/files/files/form/i-539instr.pdf). If biometrics (fingerprint) are required, the total fee is $375.
The U.S. government keeps track of individuals who are issued B-1 visas and admitted to the United States in the B-1 visa category. However, neither the U.S. State Department nor the U.S. Department of Homeland Security breaks down that information with respect to the particular B-1 subclasses that authorize employment. For example, from available information, it is impossible to know the number of domestic workers or trainees who have received B-1 visas. There is no information on where they come from, or their age, or gender. Likewise, there is no available data on the number of individuals with B-1 in lieu of H-1B visas, or other demographic information. Because the U.S. Department of Labor has no role in either of these three B-1 subclasses, that agency does not have any data pertaining to the program either. Furthermore, no federal agency publishes information about employers of B-1 workers.
A. Number of B-1 Visitors in the U.S.
The U.S. Department of Homeland Security has two sub agencies that may be involved in the B-1 program and thus may be sources of data. At the border or port of entry, the U.S. Customs and Border Patrol (CBP) interviews workers who have received B-1 visas, decides whether to grant their admission, and issues the electronic I-94. These admissions are tracked and data is published annually. Unlike certain other nonimmigrant work visa programs, the U.S. Citizenship and Immigration Services (USCIS) has no role prior to the issuance of a B-1 visa. USCIS is involved, however, when the B-1 domestic worker applies for her Employment Authorization Document after her arrival, or when any B-1 visa holder applies for an extension of the visa. While USCIS publishes information on EAD applications and extensions generally, the information is not disaggregated by nonimmigrant visa category.
The Philippines was the largest sending country for B-1 visas in 2012, followed by Mexico, Venezuela, Brazil and Cuba.99 However, because the B-1 visa is not broken down into subclasses, this information tells us nothing about the nationality of individuals who are working with B-1 visas.
Based on admissions flow, the leading sending countries are Mexico, China, India, Brazil and South Korea.100 However, in any case, because the B-1 visa is not broken down into subclasses, this information tells us nothing about the nationality of individuals who are working with B-1 visas.
93. U.S. State Department, FY 1997-2012 NIV Detail Table, available at http://travel.state.gov/visa/statistics/nivstats/nivstats_4582.html (June 2013).
94. U.S. Department of Homeland Security, Nonimmigrant Admissions by Class of Admission and Country of Citizenship: Fiscal Year 2012 (Supplemental Table 1), available at http://www.dhs.gov/yearbook-immigration-statistics-2012-nonimmigrant-adm... (September 2013).
96. Email correspondence with Office of Immigration Statistics employees, U.S. Department of Homeland Security (2013).
99. U.S. State Department, FY 1997-2012 NIV Detail Table, available at http://travel.state.gov/visa/statistics/nivstats/nivstats_4582.html (June 2013).
100. U.S. Department of Homeland Security, Nonimmigrant Admissions by Class of Admission and Country of Citizenship: Fiscal Year 2012 (Supplemental Table 1), available at http://www.dhs.gov/yearbook-immigration-statistics-2012-nonimmigrant-adm... (September 2013).
There are no regulations setting out protections for workers who are present in the United States with a B-1 visa. This makes sense because that the B-1 visa is not supposed to be for work. However, given that B-1 domestic workers, by definition, are working, there are at least some protections outlined in the U.S. State Department’s Foreign Affairs Manual (FAM).102 The FAM requires individuals applying for a B-1 visa to work in the U.S. as a domestic worker to already have a signed employment contract with their employer; this is a prerequisite for the visa.103 The required employment contract terms depend on the immigration status of the B-1 domestic worker’s employer and his or her expected duration of stay in the United States.104 Nevertheless, in all cases, the B-1 domestic worker’s contract must specify that the wage paid will be the higher of the minimum or the prevailing wage.105 The consular officer reviewing the domestic worker’s B-1 visa application is the only government official in the position to make sure that the contract exists and complies with the FAM’s requirements. While the FAM itself is not enforceable in court, the B-1 domestic worker may bring a legal action in court to enforce the employment contract’s terms if the employer does not comply with them. There are no protections in the FAM for either trainees and high-skilled workers who have a B-1 visa in lieu of H designation.
In order to qualify for a B-1 domestic worker visa, the applicant must possess an employment contract dated and signed by both the employer and the worker.107 The rules about what terms must be included vary slightly depending on whether the employer is a U.S. citizen or a nonimmigrant. U.S. citizen employers who are going to be stationed in the United States for a temporary work assignment, perhaps for a longer term, have extra requirements.
The required employment contract must guarantee that the employer will pay either the minimum or prevailing wage, whichever is greater, for an eight hour work-day.108 Moreover, the employment contract must include any other benefits “normally required for U.S. domestic workers in the area of employment.”109 Furthermore, the employer must provide at least two weeks’ notice before ending the work contract. However, the worker is not required to provide such notice of intent to leave the employment.
The fact that an individual in the United States as a B-1 trainee does not preclude treatment as an employee under the Fair Labor Standards Act (FLSA).126 Internships and training programs in the for-profit private sector almost always amount to employment under the FLSA and therefore must be paid according to its minimum wage and overtime provisions. Under the FLSA, an “employee” is “any individual employed by an employer.”127 “Employ” means “suffer or permit to work.”128 Therefore, all individuals who are “suffered or permitted” to work must be compensated for the services they provide for their employer. This definition is very broad.
102. The Foreign Affairs Manual is the organizational directive for the U.S. State Department. Chapter 9, entitled Visas provides guidance to consular officials who issue the visas but is not binding or enforceable in and of itself. See U.S. State Department, Foreign Affairs Manual, available at http://www.state.gov/m/a/dir/regs/fam/index.htm (last visited November 2013).
103. 9 FAM 41.31 N9.3.
106. The relevancy of a plaintiff’s immigration status to remedies available under substantive employment laws is nuanced and has been widely discussed elsewhere. See, eg., Kati L. Griffith, Undocumented Workers: Crossing the Borders of Immigration and Workplace Law, 21 CORNELL J.L. & PUBL. POL’Y 611, 615-26 (2012) (comprehensively outlining the “pressing need for a more integrated understanding of the sometimes complementary, sometimes conflict prone, relationship between immigration law and employment policies”); and Kati L. Griffith, U.S. Migrant Worker Law: The Interstices of Immigration Law and Labor and Employment Law, 31 COMP. LAB. L. & POL’Y J 125, 141-155 (2009) (providing background on the intersection of immigration and employment law as they pertain to migrant workers’ rights and specifically the effect of immigration status on what claims and remedies are available).
107. 9 FAM 41.31 N9.3-1 a.(5).
108. 9 FAM 41.31 N9.3-1 b; N9.3-2 b.
109. Id. The FAM does not specify the manner of determining other benefits that are normally required. However, the U.S. Department of Labor would be a source for that information.
111. 9 FAM 41.31 N9.3-2 b.
113. 9 FAM 41.31 N9.3-3 a.(4).
114. 9 FAM 41.31 N9.3-3 a.(5).
116. 29 C.F.R. § 552.102(a); see also 29 U.S.C. § 215(a)(3)(protection from retaliation).
117. 29 U.S.C. § 213(b)(21).
118. 29 C.F.R. § 552.102(b).
121. 29 C.F.R. § 785.23.
126. See, e.g., Chellen v. John Pickle Co., Inc., 344 F.Supp. 2d 1278 (N.D. Okla 2004).
127. 29 U.S.C. § 203(e)(1).
128. 29 U.S.C. § 203(g).
Because there are no worker protection regulations pertaining to B-1 visitors, there is no specific administrative enforcement scheme. There is no formal complaint procedure and no anti-retaliation protection. There is no regulatory mechanism to hold B-1 employers liable for lost wages and benefits. Because there is no specific role for the U.S. Department of Labor in the application process, its enforcement authority with regard to B-1 workers is nonexistent unless another federal law applies. State agencies customarily will have the authority to enforce any state laws that may be implicated. B-1 workers may attempt to enforce their rights in court if there is an employment contract or applicable federal or state statute allowing a private lawsuit.
The U.S. Department of Homeland Security’s (DHS) enforcement role is not directed towards B-1 worker cases in particular. However, DHS does focus some attention on trafficking, which is an issue for many domestic workers, including those working with a B-1 visa. For example, DHS runs a project known as the Blue Campaign. Designed to help combat human trafficking, the awareness campaign includes multi-lingual public service announcements, billboards, newspaper advertisements, victim assistance materials, and indicator cards for law enforcement. DHS also expanded online resources, including social media, and distributed a virtual toolkit to employers in the lodging, transportation, entertainment, agricultural, manufacturing and construction industries.
B-1 workers may file a lawsuit to enforce their rights and have their day in court in just like any other U.S. worker, as long as there is a valid claim under U.S. law and the employer is subject to U.S. courts.
B-1 workers have similar access to counsel issues as other groups of nonimmigrant workers in that lawyers may not be as willing to take their cases due to cultural differences, language barriers, and the often-short duration of work in the United States. Because B-1 domestic workers are typically low-wage earners, the amount of money owed may be small relative to the cost and complication of transnational litigation. Furthermore, because usually there is only one domestic worker per household, there is no possibility of collective representation; class action lawsuits are generally more appealing to take on because they yield higher damages awards. Foreign workers who have a B-1 in lieu of H-1B or H-3 visa may not be as vulnerable as other nonimmigrants that work in isolation or in rural areas.
134. See 45 C.F.R. Part 1611 (Financial Eligibility) and Part 1626 (Restrictions on Legal Assistance to Aliens).
135. Legal Services Corporation Program Letter 05-2 (Oct. 6, 2005), available at http://www.lsc.gov/sites/default/files/Grants/pdfs/Progltr05-2.pdf.
Government studies and advocacy reports on B-1 workers are either nonexistent or hard to find. Examining the extent of labor exploitation in the B-1 program is uncharted territory. Perhaps the most pressing issue is the fact that the U.S. government does not seem to track the use of the B-1 visa for work. The lack of basic data makes it difficult to analyze the extent to which these subclasses are utilized by businesses to fill their labor needs, not to mention the issues faced by workers. Indeed, transparency is needed to examine the potential for visa fraud as well as labor exploitation. This is especially troubling with regard to B-1 domestic workers, given documented abuse of domestic workers generally. With B-1 in lieu of H-3 trainees, the primary concern is gross underpayment of wages. With the B-1 in lieu of H-1B subclass, the primary concern stems from the fact that the workers are not paid at a level commensurate with their H-1B or American counterparts, because there are no wage requirements, and the possible misuse of the program to displace U.S. workers.
1. Case example: John Pickle Co.
The lack of any worker protections, government oversight, or transparency in the B-1 trainee program creates circumstances allowing for severe worker exploitation and even human trafficking.137 Even though B-1 visas were not designed to allow productive work in the U.S., and B-1 trainees are not supposed to be engaging in productive work for an employer, the reality is often starkly different. Any program that potentially allows work but does not require a certain wage is problematic. There is great potential for unchecked misrepresentations especially when international recruitment is involved.
The lack of government oversight and transparency in the B-1 in lieu of H-1B program creates a situation ripe for fraud. B-1 visas were not designed to allow productive work in the U.S. Indeed, the B-1 in lieu of H-1B visa is supposed to be for individuals who are already employed by the foreign company and are only in the United States for a short time. However, because it is oftentimes much cheaper to hire a foreign worker earning foreign wages, there is a great incentive to take advantage of the B-1 in lieu of H-1B program and use it to staff a company.151 Within the information technology specifically, foreign job contractors have long provided engineers to U.S. employers for foreign wages.152 In some cases, using the B-1 visa for high skilled workers is fraudulent.
137. Michael A. Scaperlanda, Human Trafficking in the Heartland: Greed, Visa Fraud, and the Saga of 53 Indian Nationals “Enslaved” by a Tulsa Company, 1 Loy. U. Chi. Int’l L. Rev. 219, 238-243 (2004) (offering a detailed explaination of how misuse of B-1 in lieu of H-3 trainee visa effectuates trafficking).
138. Chellen v. John Pickle Company, Inc., 344 F. Supp. 2d 1278 (N.D. Okla. 2004) (Chellen I).
139. Id. at 1280-81. The company had previously trained foreign workers in the United States and then used them to launch their Middle East business venture. The employer indicated that “the training program was also a “good deal” for JPC because the trainees provided “cheap labor.” While court documents apparently do not clarify whether the workers received B-1 or B-2 visas, it seems unlikely that they would have been B-2 visas because they are for tourists and by all accounts, the visa applications were clear these workers were being imported for training.
141. Id. 1280-82. The workers did not believe they were being hired for a training program and invested large sums of money and left professional jobs in India for the opportunity.
144. Id. The employer’s wife took the passports, visas, return-trip airline tickets, and I–94s and placed them in a safe.
146. Id. The U.S. employer set up separate bank accounts for each worker and the Indian recruiter deposited money into these accounts. The U.S. employer reimbursed the Indian company for payment of the workers’ salaries via a series of monthly wire-transfers set forth in a written agreement that was to be in effect for two years.
147. Several of the workers received T-visas, available for victims of human trafficking. See 8 U.S.C. § 1101(a)(15)(T). The workers did not bring claims under the Trafficking Victims Protection Act, 22 U.S.C. §§ 7101, et seq.
148. Chellen I, 344 F. Supp. 2d at 1279.
149. Chellen v. John Pickle Co., Inc., 446 F. Supp. 2d 1247, 1294 (N.D. Okla, 2006) (Chellen II).
150. Chellen I, 344 F. Supp. 2d at 1287-92 (relying on factors enumerated in Reich v. Parker Fire Protection District, 992 F.2d 1023, 1026 (10th Cir.1993) (quoting the Wage & Hour Manual (BNA) 91:416 (1975)).
151. See, e.g., D. Papademetrious and S. Yale-Loehr, Balancing Interests: Rethinking U.S. Selection of Skilled Immigrants, at 173 (1996) (“The B-1 temporary business category is generally fine in concept, as it facilitates international commerce and trade . . . . however, the B-1 in lieu of H-1B concept raises some concerns. . . . Abuses of the B-1 visa category should be controlled through better regulations and more active enforcement. . . .”).
152. See, e.g., J. Gentry and K. Kennedy-Luczak, HR how-to: foreign workers: everything you need to know about employing foreign workers, at 47 (2004) (citing “[a]buse of the ‘B-1 in lieu of H-1B policy by foreign job contractors providing computer engineers to U.S. employers under a consulting agreement”).
156. J. Preston and V. Bajaj, Indian Company Under Scrutiny Over U.S. Visas, The New York Times, (June 21, 2011) available at http://www.nytimes.com/2011/06/22/us/22infosys.html?pagewanted=all.


Oct. 12, 1984, Pub. L. 98–473, title I, § 101(h) [title VIII, § 8006], 98 Stat. 1904, 1923.
Dec. 8, 1983, Pub. L. 98–212, title VII, § 709, 97 Stat. 1439.
Dec. 21, 1982, Pub. L. 97–377, title I, § 101(c) [title VII, § 709], 96 Stat. 1833, 1851.
Dec. 29, 1981, Pub. L. 97–114, title VII, § 709, 95 Stat. 1579.
Dec. 15, 1980, Pub. L. 96–527, title VII, § 709, 94 Stat. 3081.
Dec. 21, 1979, Pub. L. 96–154, title VII, § 709, 93 Stat. 1153.
Oct. 13, 1978, Pub. L. 95–457, title VIII, § 809, 92 Stat. 1244.
Sept. 21, 1977, Pub. L. 95–111, title VIII, § 808, 91 Stat. 900.
Sept. 22, 1976, Pub. L. 94–419, title VII, § 708, 90 Stat. 1292.
Feb. 9, 1976, Pub. L. 94–212, title VII, § 708, 90 Stat. 169.
Oct. 8, 1974, Pub. L. 93–437, title VIII, § 808, 88 Stat. 1225.
Jan. 2, 1974, Pub. L. 93–238, title VII, § 708, 87 Stat. 1039.
Oct. 26, 1972, Pub. L. 92–570, title VII, § 708, 86 Stat. 1197.
Dec. 18, 1971, Pub. L. 92–204, title VII, § 708, 85 Stat. 728.
Jan. 11, 1971, Pub. L. 91–668, title VIII, § 808, 84 Stat. 2031.
Dec. 29, 1969, Pub. L. 91–171, title VI, § 608, 83 Stat. 480.
Oct. 17, 1968, Pub. L. 90–580, title V, § 507, 82 Stat. 1130.
Sept. 29, 1967, Pub. L. 90–96, title VI, § 607, 81 Stat. 242.
Oct. 15, 1966, Pub. L. 89–687, title VI, § 607, 80 Stat. 991.
Sept. 29, 1965, Pub. L. 89–213, title VI, § 607, 79 Stat. 874.
Aug. 19, 1964, Pub. L. 88–446, title V, § 507, 78 Stat. 475.
Oct. 17, 1963, Pub. L. 88–149, title V, § 507, 77 Stat. 264.
Aug. 9, 1962, Pub. L. 87–577, title V, § 507, 76 Stat. 328.
Aug. 17, 1961, Pub. L. 87–144, title II, § 201, 75 Stat. 367, 369.
July 7, 1960, Pub. L. 86–601, title II, § 201, 74 Stat. 340, 342.
Aug. 18, 1959, Pub. L. 86–166, title II, § 201, 73 Stat. 368, 370.
Aug. 22, 1958, Pub. L. 85–724, title III, § 301, title V, § 501, 72 Stat. 714, 721.
Aug. 2, 1957, Pub. L. 85–117, title III, § 301, title V, § 501, 71 Stat. 314, 321.
July 2, 1956, ch. 488, title III, § 301, title V, § 501, 70 Stat. 457, 464.
July 13, 1955, ch. 358, title III, § 301, title V, § 501, 69 Stat. 304, 312.
June 30, 1954, ch. 432, title IV, § 401, title VI, § 601, 68 Stat. 339, 347.
Aug. 1, 1953, ch. 305, title III, § 301, title V, § 501, 67 Stat. 339, 348.
July 10, 1952, ch. 630, title III, § 301, title V, § 501, 66 Stat. 520, 530.
Oct. 18, 1951, ch. 512, title III, § 301, title V, § 501, 65 Stat. 429, 443.
Sept. 6, 1950, ch. 896, Ch. X, title III, § 301, title V, § 501, 64 Stat. 735, 750.
Oct. 29, 1949, ch. 787, title III, § 301, title V, § 501, 63 Stat. 992, 1015.
June 24, 1948, ch. 632, 62 Stat. 655.
July 30, 1947, ch. 357, title I, § 1, 61 Stat. 557.
July 16, 1946, ch. 583, § 1, 60 Stat. 547, 548.
July 3, 1945, ch. 265, § 1, 59 Stat. 390.
June 28, 1944, ch. 303, § 1, 58 Stat. 580.
July 1, 1943, ch. 185, § 1, 57 Stat. 354.
July 2, 1942, ch. 477, § 1, 56 Stat. 617.
June 30, 1941, ch. 262, § 1, 55 Stat. 373.
June 13, 1940, ch. 343, § 1, 54 Stat. 358, 359.
Apr. 26, 1939, ch. 88, § 1, 53 Stat. 600.
June 11, 1938, ch. 37, § 1, 52 Stat. 649.
July 1, 1937, ch. 423, § 1, 50 Stat. 450.
May 15, 1936, ch. 404, § 1, title I, 49 Stat. 1286.
Apr. 9, 1935, ch. 54, § 1, title I, 49 Stat. 128.
Apr. 26, 1934, ch. 165, title I, 48 Stat. 621.
Mar. 4, 1933, ch. 281, title I, 47 Stat. 1577.
July 14, 1932, ch. 482, title I, 47 Stat. 670, 671.
Feb. 23, 1931, ch. 279, title I, 46 Stat. 1283, 1284.
May 28, 1930, ch. 348, title I, 46 Stat. 438.
Feb. 28, 1929, ch. 366, title I, 45 Stat. 1356.
Mar. 23, 1928, ch. 232, title I, 45 Stat. 332.
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Apr. 15, 1926, ch. 146, title I, 44 Stat. 262.
Feb. 12, 1925, ch. 225, title I, 43 Stat. 900.
1985—Pub. L. 99–145 substituted “enlistment” for “enlistments”.


text:
UKSSD Chairperson to UN – World Humanitarian Summit as Diaspora Representative – DOZ e. V.
UKSSD e. V. took part since last year 2015 in regional consultation of World Humanitarian Summit in Europe organized by Danish Refugee Council, Berghof Foundation, and African Foundation for Development – AFFORD under the name DEMAC – a project funded by EU which consists of Diaspora organizations from Syria, Sierra Leon and Somalia those based in Europe United Kingdom.
After regional consultations were concluded, UKSSD e. V. took part in The Global Consultation of World Humanitarian Summit that took place in Geneva in October 2015. UKSSD e. V. was represented by co-founder and Chairperson Mr. Abdulaziz Ramadan. UKSSD has formulated some recommendations with other Syrian Diaspora in a video message at UN center in Geneva.
In 2016, Mr. Stephan O’brien on behalf of the UN Secretary General sent an invitation to Mr. Abdulaziz Ramadan to take part at the summit in Istanbul as Diaspora Representative.
UKSSD e. V. is a youth and diaspora non-profit non-governmental organization founded in 2012 in Syria and its headquarters is based in Germany since 2014. UKSSD e. V. works in various sectors such as: Education, Child Protection, Youth Development, Peace Building, Refugees and IDPs support in conflict areas.
At the World Humanitarian Summit, UKSSD e. V. with other Diaspora actors seeks to bring more effective methods in partnership and coordination between various humanitarian actors from UN, INGOs, private sector, and member states to achieve quicker respond to crisis and translate plans into actions with other Diaspora Representatives at the summit.

matches: V. V. V. V. V. V. V.


text:
β2-microglobulin is a paradigmatic amyloidogenic protein responsible for dialysis-related amyloidosis, a disease associated to long-term hemodialyzed patients and characterized by accumulation of amyloid deposits in the osteoarticular tissues. In the early stages of amyloid fibril formation, β2-microglobulin associates into dimers and higher oligomers, but clarifications are still needed for the triggering conditions, mechanisms and specificity of dimer formation. To characterize the dimeric association process, the protein-protein interactions between three different species are investigated: namely, the native protein and the two amyloidogenic variants ΔN6 and D76N. The dimerization process is rationalized relying on state of the art computational methods. A comparative mechanism for how different mutations in the three variants can affect protein dimerization and thus fibril formation is proposed. The number of salt bridges involved at the protein-protein interface correlates with the degree of amyloidogenicity of each individual species. The findings can offer possible strategies in controlling the dimerization mechanism based on different β2-microglobulin protein mutations, which have significant roles in the fibrillogenical process.
β2-microglobulin (β2-m) pertains to the immunoglobulin family and it is the light chain of the major histocompatibility complex (MHC) class I . It is a 99 residue protein, characterized by seven anti-parallel β strands folded in a β sandwich, stabilized by a disulfide bridge between Cys25 and Cys80 (Figure 1). When released in monomeric form, β2-m loses the stability given by the interaction with MHC class I  and it can lead to Dialysis Related Amyloidosis (DRA), a systemic amyloidosis affecting patients undergoing long-term hemodialysis. In fact, biochemical studies [3–6] have reported that β2-m is the constituent of amyloid deposits of patients treated with chronic hemodialysis. The wild type β2-m requires particular conditions in order to form amyloid fibrils in vitro, as the presence of ex vivo amyloid seeds  or Cu2+ ions . Further studies on different types of tissues have shown that ΔN6, the truncated species lacking the six N-terminal (N-term) residues, is also significantly present [9–11] (around 30% of ex vivo amyloid deposits) in patients affected by DRA (Figure 1, where one-letter code sequence highlight the N-term cleavage). There are different and controversial findings regarding the mechanisms through which ΔN6 can prime the fibrillogenical behavior of the wild type [12, 16], but it is widely recognized that the truncated variant alone is able in nucleating fibrillogenesis in physiological conditions. While no other major post-translational modifications are apparently present in natural fibrillar β2-m, few years ago the first natural amyloidogenic variant of β2-m was discovered in a French family where all the heterozygous carriers of the mutation presented a multi-visceral amyloid deposit [13, 14]. The replacement of aspartic acid in position 76 with the asparagine (D76N) leads to a highly amyloidogenic protein, able to fibrillate under physiological condition  (Figure 1, where one-letter code sequence highlights the mutation position in red).
Figure 1 Backbone structure of the native β2-microglobulin (PDB entry: 1JNJ) as obtained from NMR determination  in aqueous solution.
Amyloid formation of β2-m under physiological pH conditions (around pH 7.0) has been shown to start from the fully folded native protein state . β2-m fibrillation can be triggered by conformational rearrangement of the natively folded structure [18, 19], but whether the fibrillogenical mechanism requires rearrangement to a native-like species or more highly unfolded species, or both, under different solution conditions remained unclear [4, 19–21]. Furthermore, it is not known whether the transition to the amyloidogenic intermediate may be favored by native protein dimerization or not. In the present work we rely on native conformation as a starting point to perform an enhanced conformational search of dimeric protein adducts.
Atomistic molecular dynamics is a powerful tool that can effectively complement experimental studies on this topic [23, 24]. The ability of β2-m in forming dimers under native conditions and the subsequent aggregation into tetramers has been modeled in different previous works [19, 25, 26]. In addition, dimeric units have been suggested as fundamental blocks in the construction of the mature fibrils . In this work, we perform a comparative analysis of the interactions between dimers of β2-m wild type (WT) and of amyloidogenic variants ΔN6 and D76N, starting from native conformations representative of the protein in physiological conditions. All calculations involve simulations at multiple levels spanning from docking  to conventional atomistic molecular dynamics (MD) and Temperature Replica Exchange MD (T-REMD) . The results show that the dimer interface of the WT β2-m is more unsteady than ΔN6 and D76N variant interfaces, in agreement with the enhanced amyloidogenicity of such mutants. A mechanism of sequential formation (“zipper”-like) of the salt bridges at the protein-protein interface is proposed, based on the current in silico results. The role of salt bridges in stabilizing the “zipped” dimer for each mutated species is addressed by tracking their formation during classical MD and then assessing their stability also by means of T-REMD simulations. The persistence in time of salt bridges involved at the protein-protein interface correlates with the different degree of amyloidogenicity between the compared species.
The dimeric association of wild-type β2-m or its variants in solution has been frequently observed [10, 22, 32, 33]. In order to provide a complementary approach to the interpretation of available experimental data, a preliminary docking to build protein-protein dimers is performed. We simulate dimers of WT, ΔN6 and D76N. The prediction of protein-protein encounter complexes is performed by applying the rigid-body docking as coded in SDA7 . These structures are generated by running around 5000 Brownian Dynamics (BD) simulations during which the solutes are modeled as rigid bodies in presence of implicit solvent. The adsorption free energies of the protein-protein encounter complexes obtained during the BD simulation are computed and the trajectories are clustered with the single linkage method to identify genuinely different protein orientations . Flexible refinement of selected representative structures is hence done by molecular dynamics (MD) simulations in explicit solvent. The advantage of using BD is that it mimics efficiently the physical process of diffusional association of the unbound proteins whereas the atomistic refinement is accounting for the protein conformational flexibility in MD upon association.
The same procedure is repeated for each variant: D76N variant yields one very populated complex which is representative for almost 97% of the total encounter complexes, whereas ΔN6 and WT dimers yield two different reciprocal orientations and four different orientations, respectively. Statistical data on the resultant docked complexes are reported in SI, in Table ST1. For ΔN6 we select the most populated complex (ΔN6-A) representing the 70% of the total complexes, whereas for WT the first encounter complex (WT-A) is representative of the 62%. During docking, the interaction energy between the two proteins is described by the electrostatic interaction term, UEP, dominated by the contribution of the charged side chains, and the non-polar hydrophobic interactions of the protein, Udsp. Binding between all dimeric complexes is driven mostly by non-polar hydrophobic energy terms. D76N-A dimer shows the most favorable total binding energy between all dimers. The difference between the binding energies in complexes of D76N-A and ΔN6-A is of ∼13 kT whereas between complexes of D76N-A and WT-A is of ∼9 kT, respectively. In the case of D76N-A dimer, different contact residues of each monomer forming the dimer are involved at the protein-protein binding interface: sub-unit 1 involves N-term (THR4, PRO5), BC loop (HIS31) and FG loop (THR86, SER88) and sub-unit 2 involves B strand (PHE22), CD loop (ILE46, GLU47, LYS48), D strand (GLU50, ASP53) and E strand (TYR67, GLU69) (see Figure 1 to identify strands and loops). In the case of ΔN6-A dimer the electrostatic interaction is way weaker and the dimeric interface is mainly stabilized by non-polar hydrophobic interactions. Residues involved within the binding interface for sub-unit 1 are in BC loop (SER33), DE loop (LEU54, TRP60), D strand (PHE62) and for sub-unit 2 are in BC loop (HIS31, PRO32, ASP34) and FG loop (THR86). Concerning the WT-A protein at physiological pH, the dimeric interface is stabilized mainly by non-polar hydrophobic interactions but also the electrostatic energy term partially contributed to binding. Binding patches are involving residues in N-term (LYS3), DE loop (SER61) and G strand (LYS91) for sub-unit 1 and D strand (ASP53, SER55), DE loop (PHE56, LYS58) for sub-unit 2. In the case of the WT-A, protein docking results are in agreement with NMR experimental data providing evidences of the existence of dimeric adducts in pure water with the N-term segment and DE loop involved at the dimeric interface. The DE loop and the contiguous D and E strands are involved in interfaces for all the investigated species, confirming the relevance of this portions in interactions at the beginning of the aggregation [24, 28, 35].
Starting with the most representatives dimeric complexes obtained from rigid-body BD docking, the stability of each interface is examined by using four MD runs (different by initial atomic velocities) of 400 ns each in explicit water. Given the size of the protein surface, a large number of different interactions is expected during MD, as depicted in Figure S1 in SI and here summarized in Table 1. The four independent MD runs performed on mutated dimers D76N and ΔN6 generate ensembles of conformations characterized by more extended binding interfaces with respect to the initial ones. The initial D76N-A dimer resulting from docking is able to relax into novel interface in two cases over four. Instead, ΔN6-A dimer conserves its binding patch in three cases over four. Partially this is due to the fact that population and stability of dimeric interfaces can be affected differently by relaxation during MD refinement and can also depend on mutated regions i.e. EF-loop for D76N and N-term deletion for ΔN6. In WT the binding interface is involving a smaller number of residues compared to the other mutated species.
Relaxed conformations emerging from MD show distinctive features as the formation of salt bridges in interaction interface for both D76N and ΔN6. In our findings, the extended interface between sub-units are named as “zipped” dimers, where with “zipping” mechanism we are referring to the pivotal role of a number of charged residues forming stable “salt bridge zipper” between the two monomers. During MD, a spatially sequential formation of such bridges is found. Asparagine substitution with aspartate at residue 76 increases the tendency in forming novel salt bridges within the two sub-units of D76N-A. The same capability of forming “zipped” interfaces is observed in ΔN6-A. On the contrary, formation of salt-bridges is not observed for WT-A. This is in line with the experimental evidence reporting the native protein not be able to form amyloid fibrils in vitro in physiological conditions (i.e. protein charge state at neutral pH) [12, 19, 36]. Given the different behavior of D76N and ΔN6 variants with respect to WT, we first focus on the “zipped” dimers in order to rationalize their mechanism and potential role in the formation of stable dimers, which could be relevant for early stages in fibrillation. A last section is describing briefly the “non-zipped” WT. Considering Table 1 and Figure S1 in SI, the analyzed dimers are a1 for D76N, b1 for ΔN6 and c1 for WT.
The dimerization mechanism is discussed and analyzed for the three species of β2-m, separately. The stability of our simulations is assessed by the Root Mean Square Deviation (RMSD) calculated for the protein backbone without the terminal tails and by the radius of gyration Rg (see SI, Figure S2).
In D76N dimer, the two monomers rearrange after sliding during the formation of three salt bridges (Table 2), ending in a larger number of intermolecular contacts at the interface (Figure 2a). The “salt bridge zipper” between the two monomers starts with a bond involving ASP34-ARG97 at time 84 ns followed by the formation of ARG3-GLU69 couple at 86 ns and the final slippage is assessed at 157 ns with a salt bridge between HIS84-ASP98 (Figure 3a).
At the end of simulation, the protein-protein binding interface involves the N and C-term of one monomer and leaves the terminal tails of the second monomer free to bind to another identical protein. As reported in the SI in Table ST2, the final interface relies on residues belonging to the regions identified as crucial for amyloidogenesis: more specifically, sub-unit 1 is involving residues of N-term, AB loop, C-term and of DE loop, very unstable segment of D76N β2-m variant. Also EF loop and F strand, that contains the single point mutation, are interacting with the sub-unit 2 in the final “zipped” dimer. The sub-unit 2 is involving residues belonging to complementary protein region respect to sub-unit 1. Our results are in line with previous modeling work at a more coarse grained level description , in which the authors studied dimers of D76N and put emphasis on the important role of DE loop for dimerization at physiological pH conditions, indicating as aggregation hot spots PHE56, TRP60, TYR63, ASP59, PHE62, TYR66 and also predicted as relevant residues LYS94 and TRP95. Based on a series of independent fully relaxed atomistic simulation of D76N dimers, we find a binding interface with strong presence of residues pertaining to DE loop and also residues from the C-term tail as predicted. Such interface is further supported by recent experimental evidence reporting that a specific nanobody (Nb24) is able to prevent amyloidogenesis of D76N β2-m . The effects of Nb24 binding are observed in the apical AB and EF loops and the C-terminal of D76N, directly involved in the protein self-association of the “zipped” dimer. The binding of an anti-aggregation drug or antibody with anti-amyloidogenic power in those sites would be able of blocking the protein from binding to another protein thus leading to an inhibition of the fibrillation activity, and our results confirm these experiments.
Figure 2 Reciprocal orientations before and after 400 ns of MD: on the left, initial complexes from docking; on the right, final orientations after MD. In panel a) D76N sub-unit 1 is in light blue and sub-unit 2 in light green. In panel b) ΔN6 sub-unit 1 is represented in light magenta and sub-unit 2 in gray. In panel c) WT sub-unit 1 is painted in yellow and sub-unit 2 in orange. For all systems, the N-term tail and the C-term tail are depicted in blue and red color, respectively (VMD visualization tool).
Overall, our simulations suggest that changes applied locally to D76 residue are able to highlight cooperative rearrangements on coupled regions of the protein, resembling allosteric events. In particular, when the interaction between E and F strands becomes weaker with respect to the native interaction due to the replacement of a charged residue D76 with a neutral residue N76, the strands A and B are getting more loosely bound to the protein and tend to fluctuate more freely respect to the WT, favoring the formation of a larger number of salt bridges being formed. Experimental evidences have already suggested that by weakening the interaction between D and E strand through the inclusion of mutations at Trp60 it was possible to affect the strength of the interaction between F and G strands (Trp95), inducing allosteric type behavior on the protein .
Figure 3 Zipping mechanism and salt bridges formation in dynamics of the three dimers. In each panel, the distances between charged residue and the timing of stable formation are reported. In panel a) formation of the first SB between two identical D76N monomers at 84 ns, the second at 86 ns and the final zipping at 157 ns. In panel b) lone SB formation for WT at 165 ns. In panel c) first SB formation between ΔN6 monomers at 61ns, the second at 62 ns and the final zipping at 144 ns. The last figure in panel c) shows the role of missing N-term tail (transparent blue) in allowing interaction between LYS91 and ASP34.
Once reached stability (SI, Figure S3), it is observed that initial interface involving N-terminal apical region of both sub-units change into a novel “zipped” dimer. During dynamics, larger β-sheet portions of the two sub-units (Figure 2b) play a major role. As reported with more details in SI in Table ST3, involved residues for sub-unit 1 come from BC loop, C’ strand, CD loop, D strand, F strand and from FG loop. For sub-unit 2, involved residues are from A strand, AB loop, B strand, DE loop, E-F strand, FG loop and G strand. In this case, the dimer rearrangement drives tails of sub-unit 2 toward the interface. In a previous work , Estacio et al. identified the hotspots for ΔN6 dimer aggregation in PHE30, HIS31 and HIS84. The resulting ΔN6 dimer interface in our simulations includes residues from DE loop and G strand, and also HIS84. Also in the case of ΔN6-A, we observe formation of the “salt bridge zipper” between the two sub-units: in this variant the first salt bridge is formed between residues LYS91 of sub-unit 1 and ASP34 of sub-unit 2. The “zipper”-like mechanism is plotted in Figure 3c. From Table 2, we can see that the formation of this first salt bridge occurs earlier than in any other investigated variants, and in the end it involves a larger number of salt-bridges with respect to the other variants. In fact, the bond between LYS91 of sub-unit 1 and ASP34 of sub-unit 2 occurs at time 61 ns, followed by formation of a second salt-bridge between HIS51 of sub-unit 1 and ASP59 of sub-unit 2 at time 62 ns. After some time to relax the neighboring residues, the closure occurs at time 142 ns and 144 ns by the formation of salt-bridges between residues GLU36 of sub-unit 1 with ILE7 of sub-unit 2 and GLU44 of sub-unit 1 with ARG12 of sub-unit 2. The first electrostatic contact, by which “zipping” mechanism originates, is not observed in any other variant since it is possible thanks to the missing N-term, as depicted in the zoomed structure in Figure 3c. In order to assess the role of ASP34 in the formation of the “zipped” interface, we run a MD simulation starting from dimer ΔN6 in the conformation with the first salt bridge already formed and then mutation D34N is inserted. After 180 ns, the interaction interface is involving BC loop, C strand, CD loop and FG loop for the first sub-unit where there is the mutation. The second sub-unit is involving FG loop, G strand and C terminus, and also few residues from A strand, B strand and DE loop. The simulation shows that, replacing ASP34 with ANS34, complete “zipping” mechanism does not occur (SI, Figure S12).
Sub-units of WT-A (SI, Figure S4) are able to rearrange but formation of a “salt bridge zipper” is not present as in other variants (Figure 2c). Very likely, the N-tail does not allow any initial contact between charged residues. New dimeric interaction patches (more details in SI, Table ST4) involve AB loop, EF loop, FG loop and most of the strands (A, B, C and F) for sub-unit 1 and AB, CD loops and B, E strands and C-terminal. We observe that, at the beginning of MD, both the monomers involve DE loop in the binding interface, but during dynamics they loose reciprocal interaction occuring via such loop. In fact, this portion of the protein is strongly mobile in WT and binding is observed to be unstable due to the presence of TRP60. Rennella and co-workers  indicated BC loop highly reactive in dimerization process for the wild type and then listed several relevant residues: GLN2, ARG3, LYS6 (N-term); SER28, PHE30, SER33, ASP34, GLU36 (BC loop, C strand); LEU54, SER55 (DE loop); VAL85 (FG loop); ASP96 (G strand). Comparing final residues at the interface of WT-A, we find strong participation of N-term, B strand and D strand residues for the initial docking pose, then the interface proposed by other predictions has been lost, maintaining few residues of A strand and C term. For WT, we observe the formation of a lone salt bridge between LYS75 and GLU50 at 165 ns (Figure 3b). Such bond is indeed well conserved during the entire length of the simulation but no additional salt bridges form. The interaction of the involved charged residues  highlighted GLU50 as really important residue in D-E strand strand interaction in driving anti-parallel rearrangement, while LYS75 in EF loop is very active in the dimer formation as it is more solvent accessible.
In order to rationalize the dimerization mechanisms, it is crucial to analyze each factor that may confer enhanced amyloidogenic propensity to β2-m variants. Changes in hydrophobicity and in secondary structure of the protein are the most important factors . In this section we discuss how the formation of a dimeric interfaces is accompanied by changes of these two features of protein aggregates. The variation of hydrophobic Solvent Accessible Surface Area (SASA) is studied to investigate the role of hydrophobic forces in driving dimeric rearrangement during relaxation of MD simulations. Analyzing the three dimers (as reported in plot of SASA in SI, Figures S5 and Table ST5), we observe that the formation of D76N zipped dimers is accompanied by a larger variation of hydrophobic SASA, with a decrease of around 6 nm2. On the contrary, ΔN6 and WT dimerization processes are paired with even lower variation of the hydrophobic SASA, showing an almost constant behavior of hydrophobic forces that are not contributing significantly in the overall rearrangement. For the cleaved variant ΔN6, the new configuration tends to favor the interaction between N-term charged residues in both proteins and it is stabilized mainly by favorable electrostatic forces acting in a small N-term region. It results in a variation of 2 nm2 for SASA. A similar behavior is observed for the WT dimer: the sliding between monomers is modest because of the lack of a zipping mechanism hampered by the presence of a flipping tail at N-term. In fact, the last residues of N-term are avoiding the establishment of initial stable electrostatic contacts between the two sub-units. As conformational rearrangements are suspected to be related to the transition towards fibers, we check the structural stability of the portions of each sub-units in every dimers (see SI in Figures S8, S9 and S10). In particular, for β2-m it is conceivable that very limited conformational changes may be sufficient in promoting fibrillogenesis, without strong loop rearrangement and loss of secondary structure . In D76N-A, AB and EF loops present the highest mobility with respect to other portions of the protein but, in general, the RMSD of D76N-A does not deviate from NMR structure 1JNJ. In ΔN6-A, the “zipped” dimer presents a high stability for each portion, although the most interesting rearrangement involves loop AB. Almost contemporarily in both sub-units at time 180 ns (Figure 4b) in sub-unit 1 there is a complete opening toward the solvent, while in sub-unit 2 the new orientation is toward sub-unit 1. AB loop is a quite interesting portion as it is an interaction site between MHC complex and β2-m . It is also relevant that the AB loop of sub-unit 1, which is in direct contact with the surrounding solvent, is experiencing a stronger Root Mean Square Fluctuation (RMSF) of the involved residues with respect to the sub-unit 2 (Figure 4a). A possible explanation for AB loops opening could be found in the closure of the dimer, which through “zipping” is inducing a structural rigidity of those portions of the proteins involved at the interfacial region. To compensate such effect, other portions of the dimer not directly involved in binding might become softer and more mobile, resembling allosteric perturbations observed in the NMR experiments .
Figure 4 ΔN6 dimer structural rearrangements. In panel a) RMSF for loop AB in sub-unit 1 and sub-unit 2; in panel b) RMSD for loop AB in ΔN6 dimer sub-units and monomer.
T-REMD is used to allow a more extensive search of dimeric conformational minima in the Free Energy Surface. We employ a total of 32 replicas, covering a temperature range 290–320 K [48, 50]. The adopted protocol yields an aggregated simulation time of 640 ns. The convergence of the simulation is assessed with stability of protein-protein binding interface which is not changing during the entire length of T-REMD for all performed simulations. A deeper analysis involving existence of salt bridges at the interface can be interpreted as the mechanism by which the interface is stabilized.
The formed salt bridges are tested by proving stability of the bonding. T-REMD simulations of 20 ns are performed starting from the D76N-A and ΔN6-A “zipped” dimers. Since for WT-A dimer no formation of “salt bridge zipper” is observed, we skip further simulation of this specie. The existence in time of salt bridges at interface is obtained following the replica at the lowest temperature during T-REMD. The interaction patch of dimer D76N-A is shown in Figure 5a: two out of three salt bridges are found to be stable, having a residue-residue distance of 4 Å conserved during the entire simulation (see Table 3). For ΔN6-A, two out of four salt bridges are well conserved during 20 ns of T-REMD. In fact, the distance between charged residues involved in binding interface remains constant between LYS91-ASP34 and GLU36-ILE7, as showed in Figure 5b (black and green lines, respectively). The two remaining salt bridges are experiencing a continuous breaking and reforming, enabling sub-units to slightly rotate around the conserved bonds and change the interaction interface. Nevertheless, conformational rearrangements experienced in the MD are well conserved also in T-REMD simulation, and there are no portions of the protein undergoing relevant structural changes.
Figure 5 First most populated cluster after 20 ns of T-REMD. Residues involved in SB formation are represented with van der Waals radius. In panel a) the formation of three SB within D76N dimers is depicted (left); the SB bond distance is conserved during the entire simulation (right). In panel b) four SB within ΔN6 dimer are highlighted (left); during T-REMD, only two out of four SB are conserved at the end of the simulation (right).
In this work we propose a comparative mechanism to explain how different mutations of the highly amyloidogenic D76N and ΔN6 β2-m variants can affect dimerization, characterizing intermolecular salt bridges formation. We find in particular that the number of salt bridges involved at the protein-protein interface is directly related to the degree of amyloidogenicity of each individual species (D76N > ΔN6 > WT). Our results show that only D76N and ΔN6 are able to initiate formation of salt bridges between neighboring proteins leading to a “zipping-like” mechanism. On the contrary, for WT formation of a single salt bridge is observed.
Our MD results provide new ideas on the mechanism of formation and stabilization of dimers, suggesting new experiments in the field and contributing in the clarification of how low-weight oligomers are formed in solution and how fibrillogenesis sets up.
Three proteins are considered: wild-type β2-m, ΔN6 and D76N variants. MD simulations start from the same NMR structure (PDB:1JNJ)  upon inclusion of variations: ΔN6 is obtained by cleaving the first six residues and D76N substituting residue ASP76 with ASN. In the case of D76N, the protein is relaxed before docking and a structural sampling in solution is performed for 20 ns of T-REMD, in order to include the possibile conformational changes of the mutated residue at position 76. RMSD comparison with a plethora of other available PDB structures is reported in Table SF1 in SI, with an average RMSD value below 2.8 Å for 1JNJ (PDB entries: 5CS7, 2YXF, 2D4F, 2XKS for WT; 2XKU for ΔN6; 5CSB, 4FXL for D76N). For completeness, a comparison between structures resulting from dimeric encounter complexes of WT and structures of the covalent disulphide-linked homodimer in the PDB structures (entries: 4R9H, 4RA3, 3TM6) is performed. The X-ray structures show an involvement of residues in BC loop and D strand, in agreement with docking results of WT (SI, Table ST1), indicating the importance of those regions in initial steps of oligomerization. A larger variability of dimeric interface in docking and MD simulations is expected due to the presence of a larger number of degrees of freedom involved in the association process between monomers. The three variants are protonated with the aid of the server H++ (http://biophysics.cs.vt.edu/H++) at a pH = 7.0 in order to be close to physiological conditions, resulting in a net charge of -2 for WT, +2 for D76N and -3 for ΔN6. To simulate a neutral system, we add the proper amount of Na or Cl ions for monomers and dimers of each species. Furthermore, the presence of the disulfide bridge between Cys25 and Cys80 is modeled by GROMACS.
A preliminary docking to build protein dimers is performed. Rigid-body docking method implemented in SDA 7.2  are applied to predict dimeric interfaces of encounter complexes. During the simulations, translational and rotational motions are simulated for one protein with respect to the position of another identical protein. This procedure is performed individually for WT and each mutants. In SDA software, every trajectory begins with two proteins at center-to-center distance b, and stops when a protein reaches center-to-center distance c with a larger value than b. Here, b is set to 100 Å and c to 200 Å, and the temperature is set to 300 K. The reaction criterion defines the encounter event and here we consider a successful complex to be formed when two independent contacts come within 5.5 Å. In such procedure, performed individually for the WT and for each of the mutants, the contacts should be between interface residues predefined by SDA. The diffusion coefficients are set to 0.0135 Å2/ps and 3.92 10-5 radian2/ps for the relative translational coefficient and the rotational coefficient of each protein, respectively. The time step is set to 1.0 ps for center-to-center distances up to 50 Å, and for larger distances it increases linearly. For calculation of hydrophobic desolvation energy/forces it is considered a parameter value of -0.013 kcal/mol/Å2 and for the electrostatic desolvation energy/forces a parameter value of 1.67 kcal/mol/Å2, according to Elcock et al. . Ionic strenght is set to 50 mM. We apply a single-linkage clustering method based on CA atoms and characterized by an RMSD of 3.0 Å in order to provide the smallest number of physically distinct orientations of one protein relative to the position of the other. All results given in the manuscript start from such configurations.
After performing initial docking, stability of encounter complexes is reached by running atomistic simulations for both dimers and monomers in water (the latter used as a benchmark) using GROMACS (v4.6.7). Simulations are based on the OPLS-AA force field with the SPC/E water model as implemented in the GROMACS package. The electrostatic potential, in its long-range part, is treated with particle-mesh-Ewald (PME) protocol. The radius for electrostatic interactions is set to 1.10 nm, while the long range radius is set to 1.00 nm. The simulations are performed using periodic boundary condition (PBC) in all directions. The bond lengths are treated like fixed constraints and a time step of 0.002 ps for numerical integration of the equations of motions is used. A simulation protocol chosen in our group’s previous works  is adopted: it includes an initial energy minimization using steepest descent algorithm, followed by first equilibration simulation of 500 ps under constant volume at 300 K (temperature coupling: velocity rescale ) and subsequently an equilibration simulation of 500 ps with a constant pressure of 1 bar (pressure coupling: Parrinello-Rahman ) is carried out. Finally, the stability of selected complexes is examined by running standard MD simulations at 300 K of a length of 200 ns for monomers and of 400 ns for dimers. Simulations are repeated four times using a different seed for starting velocity distribution to improve statistics. The trajectories are analyzed following a standard analysis and using tools within the GROMACS package.
Due to the intrinsically limited time scale accessible to classical MD, the parallel tempering in the form of replica exchange molecular dynamics (REMD) is also performed, following previous protocol simulation of our group [48, 50]; in particular, such simulations are applied to dimers exhibiting the most extended dimeric interface (D76N and ΔN6). REMD involves multiple independent simulations at different temperatures (T-REMD), during which running replicas periodically attempt an exchange in temperature space [51, 53]. Replicas at low temperature have an opportunity to exchange to a higher temperature where energy barriers may be more easily crossed, enhancing the effective sampling space. In fact, one of the methodological advantages of T-REMD is that a protein can explore different folding pathways at the same time and can overcome barriers or trapped states at higher temperatures. For the investigated systems, 20 ns simulations on a total of 32 replicas are employed, covering the temperature range between 290 K and 320 K . The exchange rate is 0.2 and the time interval each replica spends in a given temperature is 10 ps. Further details are reported in SI in Figure S11.
MCM, GB and SC acknowledge the CINECA award under the ISCRA initiative, for the availability of high performance computing resources and support. Oak Ridge National Laboratory is acknowledged for the supercomputing project CNMS2018-338, through the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. Facilities of the National Energy Research Scientific Computing Center (NERSC), which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231, are also acknowledged.
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Maria Celeste Maschio is a PhD student at the University of Modena and Reggio Emilia, Italy. She is working on a project about the modeling of an amyloidogenic protein in solution and on nanostructures, using classical molecular dynamics, under the supervision of Prof. Stefano Corni and Giorgia Brancolini. She spent a period of six months in King’s College London (UK) working with Prof. Carla Molteni on enhanced sampling methods as Metadynamics.
Giorgia Brancolini received her Ph.D. in Chemistry from the University of Modena and Reggio Emilia, Italy, in cooperation with the University of Ulm, Germany. She was a post-doctoral fellow at Politecnico di Milano from 2002–2004 in the Dept. of Chemical Engineering. From 2004–2010 she was a senior post-doctoral fellow at National Research Council in Italy. Since 2010 Dr. Brancolini is a fixed term research scientist at the Center S3 CNR-NANO of the Institute of Nanoscience of the National Research Council in Modena, Italy. She is working on multi scale simulations, spanning from ab initio to classical and coarse grained models, of the interactions between proteins and inorganic surfaces/nanoparticles.
Stefano Corni is full-professor of Physical Chemistry at the Dept. of Chemical Sciences, University of Padova, Italy, and a research associate with CNR-NANO Modena, Italy. He got his Ph.D. in Chemistry from the Scuola Normale Superiore di Pisa, Italy, in 2003. He is working on the classical and quantum mechanical modeling of the interactions between proteins and inorganic surfaces/nanoparticles, on the optical properties of molecules interacting with metal nanoparticles and on electron transfer in biomolecules and between biomolecules and electrodes.
Journal of Self-Assembly and Molecular Electronics, Vol. 6_1, 35–60.

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